Drosophila gene families: G-protein coupled receptors

Drosophila GPCRs have been classified into four families: rhodopsin-like; secretin-like; metabotropic glutamate-like; and atypical 7 TM proteins. This classification is based on primary and secondary structure predictions, sequence analysis using profile hidden Markov models, and sequence homology searches using BLAST. Despite the greater number and diversity of GPCRs in vertebrates and C. elegans as compared with Drosophila, the data point to conservation of hormone and neurotransmitter receptors across phyla, suggesting ancient evolutionary origins (Brody, 2000).

The rhodopsin-like family encompasses receptors for a large variety of stimuli, such as biogenic amine neurotransmitters, neuropeptides, peptide hormones, light, nucleotides, prostaglandins, leukotrienes, chemotactic peptides, and chemokines. Although their ligands vary considerably in structure, the rhodopsin-like GPCRs show sequence conservation within their seven putative TM domains (Brody, 2000).

The Drosophila photopigments form three subgroups: (1) Rh1, Rh2, and Rh6 are related to long wavelength-absorbing invertebrate visual pigments; (2) Rh3, Rh4, and Rh5 belong to a group of short wavelength-absorbing invertebrate visual pigments; (3) CG5648, which is a newly identified Drosophila opsin. Subgroups 1 and 2 are more closely related to each other than to CG5648. Drosophila opsins are quite distinct from vertebrate opsins and are more closely related to other insect and mollusk opsins and to melanopsin, a dermal opsin from Xenopus laevis. This level of sequence homology suggests that invertebrate opsins and melanopsin may share a common functional basis and evolutionary origin. Functionally, vertebrate retinal opsins require reisomerization into the 11-cis isomer, whereas invertebrate photopigments retain a covalently linked chromophore (Brody, 2000 and references therein).

GPCRs for biogenic amines, related compounds, and purines constitute a large group of receptors for classical neurotransmitters and neuromodulators that may share a common evolutionary ancestor and are present in vertebrate and invertebrate lineages. Of the 21 receptors identified in this group, 11 are described in this study. The biogenic amine GPCRs share high levels of sequence similarity within species and across phyla. Therefore, many of the newly described biogenic amine GPCRs cannot easily be classified into subgroups as defined by their putative ligands. Furthermore, it has been suggested that these receptors have changed their substrate specificities during evolution (Brody, 2000 and references therein).

Insects, and Drosophila in particular, have proven to be ideal experimental organisms for the study of the roles of biogenic amine signaling in development, learning, and addiction. Serotonin (5-HT) is involved in circadian rhythms, locomotion, feeding, learning, and memory in invertebrates. The 5-HT₂ receptor is known to play an early role in coordinating cell movements during gastrulation in Drosophila. Dopamine plays a role in the responses of Drosophila to nicotine and ethanol. Targeted expression of either stimulatory or inhibitory G-alpha subunits in dopaminergic and serotoninergic neurons blocks behavioral sensitization to repeated cocaine exposures. Octopamine and tyramine are monoamines thus far identified in arthropods and mollusks (see Tyramine β hydroxylase). Octopamine has been implicated in the establishment of associative learning in the honeybee and tyramine is essential for sensitization to cocaine in Drosophila. Drosophila receptors for most biogenic amines, with the exception of histamine, have been identified. In fact, no histamine receptors have been cloned from invertebrates. However, histamine is thought to be the neurotransmitter for Drosophila photoreceptors. Therefore, one or more of the unclassifiable biogenic amine receptors may serve the function of histamine receptor. There is a large amount of evidence supporting the existence of purinergic transmission in invertebrates, but their receptors have not been cloned to date. The newly identified gene CG9753 encodes a receptor that shares homology with vertebrate adenosine receptors and may constitute the first invertebrate purinergic GPCR (Brody, 2000 and references therein).

Twenty-five putative peptide GPCRs have now been identified, 18 of which represent newly discovered genes. The Drosophila peptide GPCRs were assigned to nine different ligand types. Approximately 30 different types of peptide GPCRs have been identified in vertebrates. Thus, there appears to be a paucity of peptide receptor types in Drosophila , suggesting that there will be fewer cognate peptide hormones in Drosophila than in vertebrates. Drosophila peptide GPCRs also appear to be more closely related to vertebrate than to C. elegans peptide GPCRs. This finding is surprising given the extensive differences between insects and vertebrates in growth and hormonal regulation (Brody, 2000).

Sequence analyses of the novel putative Drosophila peptide GPCRs suggest roles for them in regulation of growth, fluid balance, visceral functions, and sexual development. Allatostatin is a 15-amino acid insect neuropeptide that inhibits juvenile hormone synthesis. The receptors for LH, FSH, and TSH belong to a family of GPCRs characterized by large NH₂-terminal extracellular domains containing leucine repeats, which are important for interaction with glycoprotein ligands. A mutant phenotype is known for only one Drosophila peptide GPCR: the rickets mutation, which leads to developmental defects and suggests a role for this receptor in limb development. The gene rickets (rk) bears homology to vertebrate leucine-rich repeat-containing GPCRs. Another putative hormone receptor gene, CG6111, encodes a protein related to mammalian vasopressin receptors. Three novel Drosophila genes code for putative growth hormone secretagogue (GHS) receptors: CG8784, CG8795 (two closely related genes located in tandem on opposite strands of chromosome 3R), and CG9918. The vertebrate GHS receptors are involved in regulation of growth hormone release and their endogenous ligand is unknown. The presence of GHS-like receptors in Drosophila is provocative and should help to elucidate the identity of their ligands and the functions of their vertebrate homologs (Brody, 2000).

Fourteen Drosophila GPCRs, 12 of which are newly described in this study, did not show significant sequence homology to functionally characterized receptors and were included in the orphan receptor group. Most of these orphan GPCRs show higher degrees of sequence identity to C. elegans than to vertebrate GPCRs. This could be explained because their vertebrate homologs have not yet been identified. Alternatively, these orphan GPCRs may play developmental or physiological roles common to both C. elegans and Drosophila (Brody, 2000).

The secretin-like family includes receptors for many hormones such as secretin, calcitonin, vasoactive intestinal peptide, and parathyroid hormone and related peptides. The secretin-like receptors are characterized by long NH₂-terminal domains containing five conserved cysteine residues that may form disulfide bonds and by short third cytoplasmic domains. Three novel GPCRs related to vertebrate calcitonin receptors have been identified. Calcitonin receptors are involved in the regulation of Ca²⁺ homeostasis in vertebrates. Two receptors, encoded by CG8422 and CG12370, are related to insect diuretic hormone receptors. Insect diuretic hormones are a group of peptides involved in the regulation of fluid and ion secretion. The newly identified Drosophila diuretic hormone receptors share 57% sequence identity, suggestive of a gene duplication. One novel latrophilin-like receptor gene was also identified (CG8639). Latrophilins are a heterogeneous group of Ca²⁺-independent receptors for alpha-latrotoxin, a potent presynaptic neurotoxin that stimulates massive neurotransmitter exocytosis leading to nerve terminal degeneration. The endogenous ligands for latrophilins are unknown and may be involved in control of synaptic exocytosis. Genes CG11318 and CG15556 define another subgroup in the secretin-like receptor family, coding for two novel receptors that share 41% sequence identity. These GPCRs are distantly related to the HE6 receptor, a human receptor of unknown function specifically expressed in the epididymis (Brody, 2000 and references therein).

Methuselah is a Drosophila GPCR involved in modulation of life span and stress response. The mutant line methuselah, with a heterozygous mutation in the mth gene, shows increased average life span and enhanced resistance to various forms of stress. The Methuselah receptor is also essential for normal development since flies homozygous for the mth mutation displayed pre-adult lethality. No counterparts for mth have been identified in vertebrates or C. elegans. Ten novel genes related to mth have been identified in the Drosophila genome. Methuselah is most closely related to Mth-like 2 (CG17795; 60% sequence identity). Two gene clusters were identified in this family. The genes CG17084, CG17061, and mth form a cluster on chromosome 3L. CG6530 and CG6536 are located in tandem on chromosome 2R and share 76% sequence identity at the protein level, indicating a fairly recent duplication. CG16992 and CG7674 predict truncated receptors but their classification as potential pseudogenes needs experimental confirmation. Identification of the ligands for the Methuselah-like receptors should be of major biological interest (Brody, 2000 and references therein).

The ligands for the metabotropic glutamate-like GPCRs include calcium ions and amino acid neurotransmitters glutamate and gamma-amino butyric acid (GABA). Glutamate is a major excitatory neurotransmitter in invertebrates, whereas GABA is generally released from inhibitory synaptic terminals. The metabotropic glutamate-like GPCRs are characterized by very long NH₂-terminal extracellular domains containing ~17 conserved cysteine residues that may form disulfide bonds. Eight members of the metabotropic glutamate receptor-like family were identified in the Drosophila genome; seven of them are described in this study for the first time. The novel metabotropic glutamate and GABA-B receptor-like genes show very high degrees of sequence conservation with their vertebrate homologs, suggesting similar roles in synaptic function (Brody, 2000).

The Frizzled-like proteins, Starry night (Flamingo) and Bride of sevenless, are defined here as atypical 7 TM proteins, a group of receptors that share the typical topology of GPCRs but show no sequence conservation with members of the other GPCR families. These receptors are involved in tissue polarity and cell-cell signaling but their signal transduction pathways are unclear. However, there is evidence that a rat homolog of the Frizzled-like group couples to G proteins. A novel atypical 7 TM protein gene, CG4626, was identified that encodes a Frizzled-like protein, which is more closely related to mammalian Frizzled 4 than to other Drosophila Frizzled-like proteins (Brody, 2000).

The Rhomboid-like proteins can also be classifed as atypical 7 TM proteins. Three sequences from the Berkeley Drosophila Genome Project database were identified that exhibit high similarity to rhomboid. These three were named rhomboid-2 (CG12083), roughoid (CG1214), and rhomboid-4 (CG1697). Both rhomboid-2 and rhomboid-3 are cytologically located very close to the rhomboid-1 (rhomboid) gene on the third chromosome, whereas rhomboid-4 (CG1697) has been mapped to position 10C on the X chromosome by polytene chromosome in situ hybridization. Full length cDNAs were isolated for each of the new genes and their sequences were compared. The most highly conserved region spans the seven transmembrane domains; the hydrophilic amino terminus is strikingly divergent. This pattern of similarity is very like that between Drosophila rhomboid-1 and its recently identified mammalian homologs (Pascall, 1998), and suggests that the transmembrane domains provide a core function for Rhomboid-like proteins. A phylogenetic tree derived from these sequences indicates that rhomboid-3 is most closely related to rhomboid-1, followed by rhomboid-2; rhomboid-4 is the least related. The amino-terminal region of Rhomboid-4 contains two tandemly arranged EF-hand motifs that are putative calcium-binding domains. There are three further rhomboid-like genes predicted (rhomboid-5, rhomboid-6, and rhomboid-7). Rhomboid-5 (CG5364) is located at 31C; Rhomboid-6 (CG17212) at 33C, and Rhomboid-7 (CG8972) at 48E. The most conserved region encompasses the transmembrane domains, while diverging in the hydrophilic amino termini. This striking conservation of rhomboid-like genes suggests that the primordial function of these proteins is a fundamental cellular process. The restriction of Drosophila Rhomboid-1 and Rhomboid-3 function to Egfr signaling presumably represents a specialization of this original function (Wasserman, 2000).

Starry night (Stan) is a complex protein containing 7 TM domains and several cadherin, EGF-like, and laminin G domains. The stan gene may have evolved from the combination of ancestral genes coding for a secretin-like GPCR and a cell adhesion molecule. In Drosophila, Stan is implicated in establishment of tissue polarity. A novel atypical 7 TM protein that may be distantly related to secretin-like GPCRs is encoded by CG20776, which contains multiple TM domains and several leucine-rich repeats thought to be involved in protein-protein interactions. Bride of sevenless (Boss) is another atypical 7 TM protein that might be distantly related to the metabotropic glutamate-like GPCRs (Brody, 2000).

In conclusion, GPCRs constitute a very large superfamily of proteins that play a central role in eukaryotic signal transduction. The families of typical GPCRs include the rhodopsin-like, secretin-like, and metabotropic glutamate-like receptors, fungal mating pheromone, Dictyostelium cAMP receptors, and C. elegans chemoreceptors. Additionally, there are four putative (or atypical) GPCR families: the Frizzled-like receptors Rhomboid-like proteins and Drosophila olfactory and putative taste receptors. All the different GPCR families share the same seven membrane-spanning domain topology. The evolutionary relationship between the different families is uncertain since there are no significant degrees of sequence similarity between them. It is likely that they have evolved independently and convergently adopted the G protein signal transduction pathway (Brody, 2000).

Most of the 100 Drosophila or more GPCRs present in show a high degree of sequence conservation with vertebrate GPCRs. Only eight Drosophila GPCRs appear to be more closely related to C. elegans that to vertebrate receptors. There has been a large expansion and diversification of chemoreceptors in C. elegans. There is also evidence of an expansion of the peptide receptors in vertebrates and odorant receptors in mammals. Drosophila GPCRs have not expanded to a similar degree: in particular there appears to be a lower number of peptide receptors than expected. This is somewhat surprising, since it has been suggested that peptide transmitters predate biogenic amines in evolution. In C. elegans, the expansion of GPCR genes is mirrored by an expansion in G protein subunits: 20 alpha-, 2 ß-, and 2 gamma-subunit genes have been identified in the C. elegans genome (Bargmann, 1998). In contrast, the Drosophila genome contains only 6 alpha-, 3 ß-, and 2 gamma-subunit genes (Brody, 2000 and references therein).

The organization of the GPCR genes in Drosophila genome shows several differences from that found in other eukaryotic genomes analyzed to date. GPCR genes form large clusters in the genomes of C. elegans and mammals. In contrast, only small clusters of GPCR genes were identified in the Drosophila genome: six consisting of two genes and one of three genes. Substantial proportions of the vertebrate GPCR genes are thought to be intronless, but only 5 out of the 100 Drosophila GPCR genes are predicted to be intronless. The C. elegans and mammalian genomes contain a large number of GPCR pseudogenes. Only eight genes were identified in the Drosophila genome that appear to code for incomplete GPCRs, but their identity as pseudogenes will require further experimental investigation (Brody, 2000 and references therein).

Now that the full repertoire of Drosophila GPCRs is known, the next step is to match the newly identified receptors with their cognate ligands and biological functions. Systematic mutation of the Drosophila GPCRs will help determine their roles in development, neural function, and behavior and may also yield insights into the functions and mutational pathologies of their vertebrate homologs. For example, it is becoming clear that substantial overlap exists in the biological components of addiction in vertebrates and flies; consequently Drosophila should prove invaluable as a model for the study of addiction. Although it has served as a model organism for nearly a century, Drosophila has now been cast in a new role, which should further the investigation of the mechanisms of development, neural function, and disease, for which the analyses of GPCRs will prove crucial (Brody, 2000).

Identification of G protein-coupled receptors for Drosophila PRXamide peptides, Cardioacceleratory peptide, corazonin, and AKH supports a theory of ligand-receptor coevolution

G-protein coupled receptors (GPCRs) are ancient, ubiquitous sensors vital to environmental and physiological signaling throughout organismal life. With the publication of the Drosophila genome, numerous 'orphan' GPCRs have become available for functional analysis. This study analyzes two groups of GPCRs predicted as receptors for peptides with a C-terminal amino acid sequence motif consisting of PRXamide (PRXa). Assuming ligand-receptor coevolution, two alternative hypotheses were constructed and tested. The insect PRXa peptides are evolutionarily related to the vertebrate peptide neuromedin U (NMU), or are related to arginine vasopressin (AVP), both of which have PRXa motifs. Seven Drosophila GPCRs related to receptors for NMU and AVP were cloned and expressed in Xenopus oocytes for functional analysis. Four Drosophila GPCRs in the NMU group (CG11475, CG8795, CG9918, CG8784) are activated by insect PRXa pyrokinins, (FXPRXamide), Cap2b-like peptides (FPRXamide), or ecdysis triggering hormones (PRXamide). Three Drosophila GPCRs in the vasopressin receptor group respond to crustacean cardioactive peptide (Cardioacceleratory peptide), corazonin, or adipokinetic hormone (AKH), none of which are PRXa peptides. These findings support a theory of coevolution for NMU and Drosophila PRXa peptides and their respective receptors (Park, 2002b).

Genes encoding Drosophila signaling peptides having PRXa C-terminal motifs were located by using BLASTP and TBLASTN searches with parameters for finding short matching sequences. Various insect PRXa peptides previously described were used for query sequences. Mature peptides were predicted by the C-terminal sequence motif PRXG(K/R): G for amidation followed by a mono- or di-basic cleavage site. N termini were predicted after the dibasic cleavage sites (K/R)(K/R) in upstream positions proximal to the PRXG(K/R) motif. A total of three genes encoding seven mature peptides were predicted. It was not possible to identify sequences similar to AVP or to the locust AVP-like insect diuretic hormone in database searches with similar search parameters as above (Park, 2002b).

The PRXa C-terminal motif is found in a number of invertebrate and vertebrate peptides. In the invertebrates, these include the PBAN-like FXPRXa motif characteristic of the pyrokinin group, FPRXa exemplified by small cardioactive peptide and CAP2b, and PRXa of Ecdysone triggering hormone. Vertebrate PRXa peptides consist of pancreatic polypeptide (36 aa with C-terminal NMLTRPRYa), AVP (NXPRXa), and NMU-25 or -8 (25 or 8 aa with C-terminal FXPRXa) (Park, 2002b).

The Drosophila genome database (www.fruitfly.org/blast/) was searched for all genes encoding peptides with C-terminal amino acid PRXa motifs and for G protein-coupled receptors likely to be activated by these ligands. The search for peptides yielded three genes: hugin (CG6371, GenBank accession no. AJ133105), cap2b-like (CG15520, capability, GenBank accession no. AF203878), and eth (CG18105; GenBank accession no. AF170922). The gene hugin encodes two peptides, referred to here as Hug and Drm-PK-2, whose C-terminal motifs are related to the insect pyrokinins. The cap2b-like gene encodes three putative peptides related to cardioacceleratory peptides (CAPs), referred to here as CAP2b-1, -2, and -3. CAP2b-1 and CAP2b-2 contain a common C-terminal motif (FPRXa), whereas the C terminus of CAP2b-3 (GLWFGPRLa) is identical to that of the diapause hormone of Lepidoptera. The peptides ETH1 and ETH2 encoded by the gene eth possess a C-terminal PRXa motif (Park, 2002b).

Analysis of the three vertebrate PRXa peptides, NMU, AVP, and pancreatic polypeptide (PP) shows that the PRXa motifs are strictly conserved in NMU and AVP, whereas that of PP is likely a consequence of converging evolution from NPY/PYY/PP family, which includes Drosophila neuropeptide F [C-terminal motif (PH)R(YF)amide]. In this fashion, the search for PRXa-activated GPCRs in Drosophila was narrowed to those related to the AVP and NMURs (Park, 2002b).

Phylogenetic analysis reveals that NMURs occur in a monophyletic clade with four Drosophila GPCRs: CG8784, CG8795, CG9918, and CG14575. Three Drosophila GPCRs homologous to AVP receptors are CG6111, CG11325 (also known as gonadotropin releasing hormone receptor), and CG10698. CG6111 is orthologous to the vasopressin/oxytocin receptor gene family (Park, 2002b).

Putative Drosophila GPCRs in the database were amplified by RT-PCR using primers based on gene predictions in the FGENESH gene finder. Conceptual translations of these genes aligned with other GPCRs present complete seven transmembrane domains. Sequences confirmed by at least two independent RT-PCR experiments revealed several polymorphic sites compared with the Celera Drosophila genomic sequences (Park, 2002b).

Oocytes injected with cRNAs for the GPCRs generated inward currents up to 2.5 μA upon activation with appropriate ligands. It is presumed that ligand-activated inward currents in these experiments result from Gq activation of phospholipase C, liberation of inositol trisphosphate, and activation of chloride current by mobilization of intracellular calcium stores (Park, 2002b).

Drosophila GPCRs in the NMUR clade were activated by PRXa peptides with various levels of sensitivity and specificity. CG14575 was the most selective within this group, responding only to CAP2b-1 (EC50 150 nM) and CAP2b-2 (EC50 230 nM), which have an identical C-terminal VFPRVamide motif. All other peptides were inactive on application at 10 μM. In contrast, CG8795 responds to a relatively wide range of ligands, including Drm-PK-2, hug, CAP2b-3, and ETH1, listed in order of decreasing potency. Drm-PK-2 and Hug appear to have highest potency, but also induce the most severe receptor desensitization. The high level of desensitization complicated efforts to produce quantitative determinations of potency for these ligands. In contrast, ETH1 and CAP2b-3 treatment produces little or no desensitization.

CG9918 and CG8784 were insensitive to most ligands applied. CG9918 responded only to the highest concentration of CAP2b-3 applied (10 μM), and was otherwise insensitive to all other ligands applied at this concentration. Similarly, CG8784 was activated only by Drm-PK-2 or Hug applied at 10 microM (Park, 2002b).

Thus Drosophila GPCRs in the NMUR group respond to the PRXa peptides, Hug, Drm-PK-2, CAP2b-1 to -3, and ETH. Non-PRXa peptides such as proctolin, FMRFamide, and diuretic hormone produced no response at 10 μM, the highest concentration tested. The range of ligand concentrations sufficient to activate each GPCR ranged from low nanomolar to micromolar. CG14575 was the most ligand-selective receptor in this group, responding only to low nanomolar concentrations of CAP2b-like peptides CAP2b1 and CAP2b-2 having FPRXa motifs, whereas CAP2b-3, a mature peptide from the same gene having FXPRXa motif had no effect on CG14575 at 10 microM (Park, 2002b).

It seems likely that CG14575 is involved in ion transport functions associated with diuresis in Drosophila. It has been shown that Drosophila CAP2b-1 and -2 act on principal cells of Malphighian tubules, stimulating fluid secretion through the calcium-nitric oxide-cGMP pathway. It will be interesting to determine whether CG14575, the putative CAP2b-1/CAP2b-2 receptor from this study, is expressed in Malpighian tubules (Park, 2002b).

CG8795 responds to a different set of nonoverlapping PRXa peptides, being most sensitive to Hug and Drm-PK-2. These peptides produce activation at low nanomolar concentrations accompanied by marked receptor desensitization, making it difficult to ascertain a reliable EC50 value for these peptides. CG8795 also shows moderate sensitivity to ETH1 and CAP2b-3, responding to mid- to high nanomolar concentrations. Interestingly, ETH2 had no effect at 10 μM. The responses of CG8795 to a wide range of peptides were unexpected. Although Drm-PK-2 was most active, Hug, ETH1, and CAP2b-3 also produced robust responses. The ligands active on this receptor also include Manduca sexta MasETH and Heliothis virescens HezPBAN at 10 microM concentration. However, some obvious selectivity was apparent, with no responses registered to CAP2b-1 and -2, ETH2, and Manduca PETH applied at 10 microM (Park, 2002b).

Activation of CG8795 by both Hug and ETH1 raises the possibility of its involvement in ecdysis. Such a possibility is indicated not only by its sensitivity to ETH1 (which is known to be obligatory for ecdysis signaling). Ecdysis deficiency is induced by ectopic expression of the hugin gene. Furthermore, the hugin gene product Hug mimics ETH1 by inducing ecdysis behavior in wild-type flies and by rescuing ecdysis deficiency in buttoned-up eth null mutants. Given that Hug and ETH1 activate both CG8795 and ecdysis behavior, several interpretations are possible. CG8795 may be involved in ecdysis signal transduction, and both Hug and ETH1 are ecdysis signaling molecules. Alternatively, CG8795 is not involved in ecdysis, but can be activated by relatively high concentrations of ETH1 acting as a Hug agonist. According to this alternative scenario, CG8795 could be involved in other physiological functions such as pheromone biosynthesis as a Hug and/or Drm-PK-2 receptor. Further work is needed to clarify an authentic role for CG8795 and function of Hug in the ecdysis signaling pathway (Park, 2002b).

The remaining GPCRs in the NMU group, CG8784 and CG9918, respond only to high levels (10 microM) of Hug and Drm-PK-2, and CAP2b-3, respectively. It is possible that the endogenous signal transduction machinery in the Xenopus oocyte is inappropriate for mediation of functional receptor activation for CG8784 and CG9918. This assay system generates a presumed calcium-activated chloride current known to be activated exclusively by Gq coupled pathways. GPCRs can be coupled to a variety of G proteins, including Gi/o, Gs, and Gq, with various degrees of efficiency and specificity. Poor coupling of heterologously expressed GPCRs to Gq in the Xenopus oocyte clearly could result in artifactually low affinity estimates. In particular, CG9918 and CG8784 were found to be largely insensitive to all ligands tested (Park, 2002b).

The functions of PRXa peptides known thus far in the vertebrates include activation of ion transport and contractile activity in intestine and arterial musculature via the NMUR. In invertebrates, functions for many of the PRXamide peptides remain uncertain, biological activity having been inferred from standard assays for visceral muscle contraction. For example, early demonstrations of activity for the pyrokinins (FXPRXa) were based on stimulation of gut, oviduct, and heart, whereas more recent data implicating them in pheromone biosynthesis and cuticle melanization are more suggestive of authentic physiological functions. The FPRXa peptides, including small cardioactive peptides and cardioacceleratory peptide (CAP2b), were isolated based on their activity in heartbeat modulation but may be involved in water and ion transport. Finally, although all other PRXa peptides are produced in the central nervous system, ETH (PRXa) is produced peripherally in epitracheal Inka cells and acts on CNS to trigger central pattern generators leading to ecdysis behavior. Knowledge of the expression patterns of the receptor GPCRs will likely provide new insights into the true physiological functions for the PRXa peptides (Park, 2002b).

Identification of Drosophila neuropeptide receptors by G protein-coupled receptors-ß-Arrestin2 interactions

Activation of G protein-coupled receptors (GPCR) leads to the recruitment of ß-arrestins (see Arrestin 2). By tagging the ß-arrestin molecule with a green fluorescent protein, the activation of GPCRs in living cells can be visualized. This approach was used to de-orphan and study 11 GPCRs for neuropeptide receptors in Drosophila. The identities of ligands for several recently de-orphaned receptors, including the receptors for the Drosophila neuropeptides proctolin (CG6986), neuropeptide F (CG1147), corazonin (CG10698), dFMRF-amide (CG2114), and allatostatin C (CG7285 and CG13702), were verified. CG6515 and CG7887 were de-orphaned by showing that these two suspected tachykinin receptor family members respond specifically to a Drosophila tachykinin neuropeptide. Additionally, the translocation assay was used to de-orphan three Drosophila receptors. CG14484, encoding a receptor related to vertebrate bombesin receptors, responds specifically to allatostatin B. Furthermore, the pair of paralogous receptors CG8985 (Myosuppressin receptor 1) and CG13803 (Myosuppressin receptor 2) responds specifically to the FMRF-amide-related peptide dromyosuppressin (Myosuppressin). To corroborate the findings on orphan receptors obtained by the translocation assay, it was shown that dromyosuppressin also stimulates GTPgammaS binding and inhibits cAMP binding by CG8985 and CG13803. Together these observations demonstrate the ß-arrestin-green fluorescent protein translocation assay is an important tool in the repertoire of strategies for ligand identification of novel G protein-coupled receptors (Johnson, 2003).

The translocation of ßarr2-GFP chimeras has been used to assess desensitization of a variety of known diverse mammalian GPCRs. To evaluate the efficacy of the translocation assay to study Drosophila GPCRs, known Drosophila peptide GPCRs that were demonstrated previously to signal through disparate pathways were tested with a panel of synthetic Drosophila peptides. HEK-293 cells transiently expressing the proctolin receptor encoded by CG6986 displayed clear translocation of the ßarr2-GFP to the membrane within 10 min of exposure to 1 µM proctolin but not to any of the other 16 Drosophila neuropeptides. Translocated GFP had the appearance of discrete puncta ranging to a continuous halo. To quantify the robust nature of the response, 100 GFP-positive cells in each of three independent transfections were scored for translocation 20 min after exposure to proctolin. In these cases, it was found that 89, 91, and 95 cells, respectively, displayed GFP translocation. In these and subsequent experiments cells were scored within 20 min of exposure to test ligands (Johnson, 2003).

The receptor for neuropeptide F (CG1147) belongs to the family of NPY-like receptors and signals through G_i-mediated pathways. Cells expressing this receptor displayed ßarr2-GFP translocation in response to its cognate ligand, NPF, at micromolar concentrations. These cells did not respond to any of the other peptides tested. Cells expressing the corazonin receptor encoded by CG10698, which is related to the vasopressin/oxytocin receptor family, displayed translocation of ßarr2-GFP to the membrane specifically in response to 1 µM corazonin. Cells expressing the dFMRF-amide receptor encoded by CG2114, which is related to the neurotensin/thyrotropin-releasing factor receptor family, displayed ßarr2-GFP translocation in response to the dFMRF-amide peptide DPKQDFMRF-amide and to the related peptide DMS, at micromolar concentrations, but not to any other peptide tested (Johnson, 2003).

Two somatostatin-like receptors (CG7285 and CG13702) in Drosophila have been shown to respond to Ast-C in Xenopus oocytes. Exposure of HEK-293 cells expressing CG7285 or CG13702 receptors to Ast-C peptide at micromolar concentrations did not produce detectable GFP translocation. Likewise, these receptors did not display any changes in intracellular calcium levels in response to Ast-C application, with or without a co-expressed promiscuous Galpha protein subunit. The C termini of these receptors have multiple clusters of serine/threonine residues that are potential targets for GRK phosphorylation. It was reasoned that this lack of a detectable response to Ast-C might be due to intrinsic phosphorylation by the endogenous complement of GRKs. In mammalian GPCRs, certain receptors are constitutively phosphorylated and associated with ß-arrestin which, at steady state, results in their trafficking to endocytic vesicles and a loss of signaling function. With these receptors, inhibiting clathrin -mediated endocytosis reverses their intracellular localization phenotype. It also re-establishes, at least in part, their signaling function. To address that possibility, CG7285 and CG13702 were each co-transfected with the dynamin K44A mutant to inhibit internalization, and potentially redistribute the receptors to the plasma membrane. Now in the presence of dynamin K44A, the association of the CG13702 receptor with ßarr2-GFP becomes apparent in the unstimulated state and is further enhanced upon addition of Ast-C. Cells expressing these receptors (with dynamin K44A) did not respond to any of the other peptides tested. Thus, blocking endocytosis can interfere with receptor trafficking but does not change the pharmacological specificity of ligand interactions (Johnson, 2003).

CG6515 and CG7887 are predicted to encode paralogous receptors that are related to the tachykinin family of receptors. Both receptors have been shown to respond to heterologous tachykinin peptide. However, neither Drosophila receptor has yet been shown to respond to native Drosophila tachykinins, and thus both remain essentially orphans. Following exposure to a putative Drosophila tachykinin peptide, translocation was observed of ßarr2-GFP in cells expressing either CG6515 or CG7887. Cells expressing either receptor did not respond to any of the other peptides tested (Johnson, 2003).

Several observations were made that indicated the possible identities of certain orphan GPCRs. CG13803 is predicted to encode a receptor related to the neurotensin/thyrotropin-releasing factor receptor family. Cells expressing CG13803 displayed translocation of ßarr2-GFP to the membrane following exposure to the neuropeptide DMS at 1 µM and at 100 nM concentrations. CG13803 cells did not respond to any other peptide tested. CG14484 is predicted to encode a receptor related to the bombesin receptor family. Translocation of ßarr2-GFP by these cells was observed in response to 1 µM Ast-B-1 neuropeptide but not to any other peptide tested (Johnson, 2003).

Observations on the CG13803 orphan receptor were extended by considering the potentiating effects of co-expressing GRK. With mammalian GPCRs, co-expression of GRKs can accelerate the kinetics and the extent of ßarr2-GFP translocation. It is thought that certain receptors may require more GRK to be effectively phosphorylated. CG8985 encodes a member of the neurotensin/thyrotropin-releasing factor receptor family and is paralogous to CG13803. Cells expressing CG8985 and overexpressing GRK2 also responded to 1 µM DMS with ßarr2-GFP translocation but not to 1 µM DPKQDFMRF-amide or any other peptide tested, under any condition. Although cells expressing CG13803 responded to DMS independently of additional GRK2 expression, this manipulation did cause a change in the response profile; CG13803 cells co-expressing GRK2 now also responded to DPKQDFMRF-amide at both 1 µM and 100 nM but not to any other peptide tested. Co-expression of GRK2 with the CG6986, CG1147, CG10698, CG6515, CG7887, or CG14484 receptors did not alter the profiles of ßarr2-GFP translocation responses of those cells to a broad range of test peptides (Johnson, 2003).

To extend the observations established with the ßarr2-GFP translocation assay, the sensitivity and selectivity of CG13803 and CG8985 for DMS and DPKQDFMRF-amide were evaluated by two additional measures of GPCR activation. CG13803 expressing cells displayed significantly higher [³⁵S]GTPgammaS binding at doses as low as 10 nM of the DMS peptide. Changes in intracellular calcium and (indirectly) for cAMP levels in response to CG13803 or CG8985 activation were also assayed. No significant increases in intracellular calcium or cAMP were elicited by exposure of either receptor to DMS or to DPKQDFMRF-amide. With cells that were exposed to forskolin, both DMS and DPKQDFMRF-amide produced significant decreases in cAMP levels suggesting that these receptors are coupled to inhibitory G proteins. Estimated EC₅₀ values for CG13803 were 0.17 nM (r² = 0.95) for DMS and 4.2 nM (r² = 0.95) for DPKQDFMRF-amide. For CG8985 cells, estimated EC₅₀ values were = 1.8 nM (r² = 0.89) for DMS and 13 nM (r² = 0.92) for DPKQDFMRF-amide. Again DMS was significantly more potent than DPKQDFMRF-amide, and that feature recapitulated results obtained with the ßarr2-GFP translocation (Johnson, 2003).

For mammalian GPCRs, it has been shown that the pattern of ßarr2-GFP translocation falls into two categories. Class A receptors maintain translocated ßarr2-GFP at the plasma membrane. Class B receptors have C-terminal clusters of serine and threonine residues and thus higher affinity for ß-arrestins; the class B receptors internalize the translocated ßarr2-GFP into endocytic vesicles. This receptor internalization is visible within 10 min post-treatment as the formation of round fluorescent vesicles that often have non-fluorescent centers. The translocation responses of cells expressing the Drosophila peptide GPCRs tend to show a similar categorization. Specifically, cells expressing CG2114, CG6515, CG6986, CG7285, CG8985, CG13702, CG13803, and CG14484 typically display class A type characteristics. Cells expressing CG1147, CG7887, and CG10698 typically display class B type characteristics (Johnson, 2003).

Unexpected role of a conserved domain in the first extracellular loop in G protein-coupled receptor trafficking

G protein-coupled receptors are the largest superfamily of cell surface receptors in the Metazoa and play critical roles in transducing extracellular signals into intracellular responses. This action is mediated through conformational changes in the receptor following ligand binding. A number of conserved motifs have critical roles in GPCR function, and this study focused on a highly conserved motif (WxFG) in extracellular loop one (EL1). A phylogenetic analysis documents the presence of the WxFG motif in approximately 90% of Class A GPCRs and the motif is represented in 17 of the 19 Class A GPCR subfamilies. Using site-directed mutagenesis, the conserved tryptophan residue was mutagenized in eight receptors which are members of disparate class A GPCR subfamilies from different taxa. The modification of the Drosophila leucokinin receptor shows that substitution of any non-aromatic amino acid for the tryptophan leads to a loss of receptor function. Additionally, leucine substitutions at this position caused similar signaling defects in the follicle-stimulating hormone receptor (FSHR), Galanin receptor (GALR1), AKH receptor (AKHR), corazonin receptor (CRZR), and muscarinic acetylcholine receptor (mACHR1). Visualization of modified receptors through the incorporation of a fluorescent tag revealed a severe reduction in plasma membrane expression, indicating aberrant trafficking of these modified receptors. Taken together, these results suggest a novel role for the WxFG motif in GPCR trafficking and receptor function (Rizzo, 2018).

Neurochemical organization of the Drosophila brain visualized by endogenously tagged neurotransmitter receptors

Neurotransmitters often have multiple receptors that induce distinct responses in receiving cells. Expression and localization of neurotransmitter receptors in individual neurons are therefore critical for understanding the operation of neural circuits. This study describes a comprehensive library of reporter strains in which a convertible T2A-GAL4 cassette is inserted into endogenous neurotransmitter receptor genes of Drosophila. Using this library, the expression of 75 neurotransmitter receptors was profiled in the brain. Cluster analysis reveals neurochemical segmentation of the brain, distinguishing higher brain centers from the rest. By recombinase-mediated cassette exchange, T2A-GAL4 was converted into split-GFP and Tango to visualize subcellular localization and activation of dopamine receptors in specific cell types. This reveals striking differences in their subcellular localization, which may underlie the distinct cellular responses to dopamine in different behavioral contexts. These resources thus provide a versatile toolkit for dissecting the cellular organization and function of neurotransmitter systems in the fly brain (Kondo, 2020).

The comprehensive collection of T2A-GAL4 lines generated in this study enabled performance of a brain-wide expression profiling of endogenous neurotransmitter receptors at high resolution. The T2A-GAL4 knockin system can faithfully recapitulate endogenous gene expression, without disrupting the function of the tagged protein in most cases (Kondo, 2020).

The configuration of T2A-GAL4 knockin system has several key features that together make it advantageous to existing methods and resources. The first is the choice of the insertion site. C-terminal insertion used in this system is less likely to disturb the endogenous expression of the target than N-terminal or internal insertion, as it does not block communication between the promoter and intronic enhancers. Most neurotransmitter receptor genes contain multiple large introns, which are known to contain important cis-regulatory elements. Second, the inserted GAL4 transgene is followed by the endogenous 3' UTR of the target gene in this system. 3' UTR sequences are known to affect gene expression by regulating mRNA stability and subcellular localization. Importantly, recent studies in Drosophila have revealed that many neuronally expressed genes have long 3' UTR sequences that play critical roles in proper expression. Thus, the T2A-GAL4 knockin system recapitulates gene expression not only at the transcriptional level but also at the post-transcriptional level. Last, recombinase sites flanking the T2A-GAL4 cassette allow replacement of T2A-GAL4 with any other reporter genes by recombinase-mediated cassette exchange (RMCE). Unidirectional RMCE in this system offers a more rapid and straightforward way of transgene replacement than other systems. A series of exchange vectors was developed for converting T2A-GAL4 lines into other transcriptional activators, fluorescent fusions, and activity reporters. This versatility of this resource allowed the characterization not only expression patterns but also the subcellular localization of dopamine receptor proteins, as well as to visualize the dynamic regulation of receptor levels and activity (Kondo, 2020).

Recently, a large-scale collection of gene-trap lines based on the MiMIC system and a T2A-GAL4 collection similar to the one described in this study have been developed. Both the resources and these collections represent versatile platforms for endogenous gene tagging. MiMIC insertions are located in coding introns and produce an internal GFP fusion. A previous study showed that ~30% of MiMIC-GFP insertions disrupt protein function and they could also disrupt subcellular localization. Although C-terminal tags are less likely to affect protein folding, they could disturb the function of certain proteins, in which the C terminus is modified or is important for interaction with other proteins. Thus, these collections complement each other in many respects, allowing users to choose reagents optimal for their studies (Kondo, 2020).

Systematic clustering analysis of neuropils identified several brain regions with characteristic expression profiles of neurotransmitter receptors. Among others, neuropils constituting the MB and the central complex were conspicuously different from the rest of the brain. This observation is in line with their unique inter-neuropil connectivity previously revealed by connectome analysis: both the MB and the central complex lack overt connectivity with the peripheral nervous system and are considered higher order integrative centers of the brain. It is speculated that the unique combinations of receptors in these centers provide the basis of complex information processing in multi-modal integration (Kondo, 2020).

At the level of individual cells, combinatorial expression of receptors could define the diverse responses of neurons to inputs from outside. Enormous complexity was observed in the expression profiles of transmitter receptors across the brain. The result of clustering analysis shows that receptors for the same ligand molecule or the same ligand type have similar expression patterns. Certain receptors expressed in the same neuron might function cooperatively by forming heterooligomers to recruit different G proteins or to form ion channels with different property. Specific functions of such receptor complexes will further contribute to the diversity of cellular responses, thereby augmenting the computational capacity of the neuronal circuit (Kondo, 2020).

All dopamine receptors were expressed in the MB despite the fact that they have clearly distinct functions in fly behavior. This study confirmed co-expression of multiple dopamine receptors in individual Kenyon cells by simultaneous visualization of two receptors. This observation corroborates the results of a recent singe-cell transcriptome analysis in the fly brain. It is, however, in stark contrast to the situation in the mammalian striatum, where D1 and D2 receptors are expressed in segregated cell populations and mediate the bidirectional dopamine inputs. In the fly, segmentation was found of the neuronal cell membrane by differential subcellular localization of receptor proteins. Furthermore, various tagging approaches of endogenous receptors revealed unexpected subcellular localization of DopEcR as well as experience-dependent regulation of protein levels and activity. It is thus proposed that differential receptor localization along the cell membrane underlies distinct subcellular responses to dopamine input in different contexts (Kondo, 2020).

Quantitative control of GPCR organization and signaling by endocytosis in epithelial morphogenesis

Tissue morphogenesis arises from controlled cell deformations in response to cellular contractility. During Drosophila gastrulation , apical activation of the actomyosin networks drives apical constriction in the invaginating mesoderm and cell-cell intercalation in the extending ectoderm. Myosin II (MyoII; Zipper) is activated by cell-surface G protein-coupled receptors (GPCRs), such as Smog and Mist , that activate G proteins, the small GTPase Rho1 , and the kinase Rok . Quantitative control over GPCR and Rho1 activation underlies differences in deformation of mesoderm and ectoderm cells. The GPCR Smog activity is concentrated on two different apical plasma membrane compartments, i.e., the surface and plasma membrane invaginations. Using fluorescence correlation spectroscopy, the surface of the plasma membrane was probed, and it was shown that Smog homo-clusters in response to its activating ligand Fog . Endocytosis of Smog is regulated by the kinase Gprk2 and beta-arrestin-2 that clears active Smog from the plasma membrane. When Fog concentration is high or endocytosis is low, Smog rearranges in homo-clusters and accumulates in plasma membrane invaginations that are hubs for Rho1 activation. Lastly, this study found higher Smog homo-cluster concentration and numerous apical plasma membrane invaginations in the mesoderm compared to the ectoderm, indicative of reduced endocytosis. Dynamic partitioning of active Smog at the surface of the plasma membrane or plasma membrane invaginations has a direct impact on Rho1 signaling. Plasma membrane invaginations accumulate high Rho1-guanosine triphosphate (GTP) suggesting they form signaling centers. Thus, Fog concentration and Smog endocytosis form coupled regulatory processes that regulate differential Rho1 and MyoII activation in the Drosophila embryo (Jha, 2018).

Tissue morphogenesis requires control over changes in cell shape and cell-cell contacts, which depend on the spatiotemporal regulation of actomyosin contractility. In Drosophila embryos, mesoderm invagination is driven by apical constriction, a geometric cell shape change facilitated by medial-apical Myosin II activation. In the ectoderm, tissue extension arises from cell-cell intercalation, whereby cells undergo neighbor exchange through the polarized remodeling of cell junctions. Junction remodeling is driven by medial-apical MyoII contractile pulses and MyoII planar polarized accumulation (Jha, 2018).

Actomyosin contractility is regulated by conserved signaling pathways. MyoII regulatory light chain is activated by Rho-kinase (Rok) downstream of the small GTPase Rho1, which in turn is regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). This conserved pathway was shown to be under the direct control of signaling at the cell surface, such as Celsr in vertebrate neural tube formation and G protein-coupled receptors (GPCRs) in early Drosophila embryos. The GPCRs Mist (Manning, 2013) and Smog (Kerridge, 2016) transduce signals from the secreted ligand Fog in the Drosophila presumptive mesoderm (Mist and Smog) and ectoderm (Smog). Medial-apical MyoII activation progresses downstream of hetero-trimeric G proteins Gα12/13, Gβ13F, and Gγ1 in both mesoderm and ectoderm. In the mesoderm, high medial-apical MyoII activation is under a stable regime that ensures persistent apical constriction, while in the ectoderm, intermediate medial-apical MyoII activation is under a pulsatile regime that enables cell-cell intercalation. Therefore, to understand how quantitative activation of MyoII is generated and its temporal dynamics encoded, it is necessary to decipher the regulation of GPCR signaling (Jha, 2018).

Differential MyoII activation in the mesoderm and ectoderm is partly imparted by the ligand Fog, co-expression of Mist and Smog in the mesoderm, as well as by the mesoderm-specific transmembrane protein T48 , which enhances apical recruitment of RhoGEF2 and, thereby, is proposed to potentiate Rho1 and MyoII activation. High Fog expression in mesoderm activates high MyoII, while in the ectoderm low Fog expression leads to low activation of MyoII. However, in general, ligand availability is one of several mechanisms impacting GPCR activation and signaling. Various cell culture studies have focused on the other modalities that regulate GPCR signaling. The major regulators of GPCR signaling are G protein-coupled receptor kinases (GRKs) that phosphorylate GPCRs and trigger signal termination, by allowing β-arrestin binding and recruitment of other adaptor proteins. In turn, β-arrestins direct activated receptors to clathrin-coated pits and remove them from the plasma membrane by endocytosis. While removal of activated GPCRs from the plasma membrane via endocytosis terminates GPCR signaling, it also reduces the number of receptors present on the surface for ligand stimulation. This effectively sets a quantitative control over GPCR signaling via endocytosis. Drosophila has only one non-visual GRK (Gprk2) and one non-visual β-arrestin-2 (kurtz). Gprk2 mutant mothers show aberrant contractility in the mesoderm lateral cells, and it was suggested that Gprk2 attenuates Fog-dependent MyoII activation in these cells. Eggs lacking Kurtz display cuticle phenotypes and suggest gastrulation defects. These data indicate that Kurtz plays a role with Gprk2 to terminate Fog signaling and could control Rho1 and MyoII via GPCR endocytosis. Its function in the mesoderm and ectoderm has not been addressed (Jha, 2018).

Conventionally, GPCR signaling from the plasma membrane is thought to occur via ligand binding and subsequent signal transduction via G proteins that relay the information to the interior of the cell. Apart from GPCR endocytosis, the localization of GPCR within the cell membrane will influence GPCR signaling. Lateral movement of GPCRs within the plasma membrane is often restricted to specific nano-domains, suggesting that selective compartmentalization is necessary for efficient signaling as it can increase GPCR localization and clustering. GPCR clustering in the form of homo- and hetero-oligomers has been reported to control both signal amplification as well as receptor recycling. Whether the main role of GPCR clustering is for chaperoning active receptors for transport or to control GPCR signaling specificity remains unclear, especially during development. To understand GPCR signaling during tissue morphogenesis, it is important to elucidate both the clustering of GPCRs at the plasma membrane and the role of endocytosis (Jha, 2018).

This study investigated the quantitative regulation of the GPCR Smog signaling by endocytosis in both the ectoderm and the mesoderm. Fog was shown to promote homo-clusters of Smog, while endocytosis rapidly removes Smog homo-clusters from the surface of the plasma membrane in the ectoderm. Dynamic partitioning of active Smog homo-clusters in two plasma membrane compartments, the surface or the plasma invaginations, was shown to directly impact Rho1 and MyoII activation. In the mesoderm, numerous apical plasma membrane invaginations and high Smog homo-clusters correlate with high Rho1 and MyoII activation compared to the ectoderm (Jha, 2018).

Epithelial cells exhibit different types of cell deformations owing to quantitative control over cell contractility that arises from contraction of the actomyosin cytoskeleton. GPCR signaling relays information conveyed by tissue-specific factors in the mesoderm and ectoderm to control this quantitative regulation during tissue morphogenesis. Rho1-dependent activation of MyoII during both apical constriction in the mesoderm and cell-cell intercalation in the ectoderm is controlled by GPCR signaling. Activation of the GPCR Smog underlies Rho1 activation in both mesoderm and ectoderm. It is believed that differential regulation of the GPCR Smog and other GPCRs underlies these tissue-specific differences in MyoII activation. This partly relies on the fact that Fog, the activating ligand, is present at higher levels in the mesoderm than in the ectoderm. This work sheds new light on this process by probing the plasma membrane organization and distribution of Smog in conditions that affect both endocytosis and production of the ligand Fog (Jha, 2018).

Probing the ectodermal cells with FCS, Smog homo-clusters on the surface of apical plasma membrane is reported and this process depends on Fog. When Fog is absent, such as in a fog-dsRNA, the brightness per Smog::GFP unit is lower, suggesting that Fog induces the formation of Smog homo-clusters. Dynamic exchange of homo-clustered Smog occurs sbetween the surface and plasma membrane invaginations. This dynamic distribution of Smog between the two plasma membrane compartments is strongly dependent upon both the rate of Smog endocytosis and Fog concentration. Increasing Fog concentration or reducing Smog endocytosis enhances the presence of Smog homo-clusters in apical plasma membrane invaginations, which results in an apparent decrease in Smog homo-clusters at the cell surface. When Fog concentration is high under conditions where Smog endocytosis is reduced, for example, when β-arrestin-2 is knocked down, Smog homo-clusters accumulate at the surface as well as in the plasma membrane invaginations. Thus, Fog concentration and Smog endocytosis form coupled regulatory processes that control the Smog cluster formation and influence the distribution of active Smog in different plasma membrane compartments. Importantly, this controls the quantitative activation of Rho1 and MyoII. Under low-endocytosis regimes (Gprk2 or β-arrestin-2 knockdowns in the ectoderm), high levels of active Rho1 accumulate in the apical plasma membrane invaginations. It is proposed that the apical plasma membrane invaginations are signaling hubs, where signaling components could concentrate, give rise to high G protein signaling (e.g., Gα12/13), and sustain high MyoII activation. The size and stability of these signaling invaginations is tuned by endocytosis, and they may provide a means to control the strength and persistence of signaling. Pulsatile active Rho1 in the ectodermal cells requires intermediate Rho1 activation. In the ectoderm, low Fog expression and rapid Smog endocytosis by Gprk2 and β-arrestin-2 lead to intermediate activation of Rho1. In turn, intermediate Rho1 activation at the apical plasma membrane creates the conditions required for self-organized actomyosin dynamics associated with pulsation (Jha, 2018).

This study also points to the possibility of tissue level regulation of endocytosis and plasma membrane compartmentalization of GPCRs. Large apical plasma membrane invaginations are observed in the mesoderm compared to the ectoderm. In the mesoderm, Smog accumulates in larger, more numerous, apical plasma membrane invaginations, and it displays larger Smog homo-clusters compared to in the ectoderm. In the mesoderm, Rho1 and MyoII activation is higher. Another GPCR, Mist produced in the mesoderm, works synergistically with Smog to boost Rho1 and MyoII activation (Manning, 2013). This is also due to the expression of another GPCR Mist in the mesoderm and to Fog being present at higher levels in the mesoderm. Ectodermal cells have similar properties of high Smog homo-clusters when Fog is overexpressed and GPCR endocytosis is slowed down. An intriguing possibility is that Smog and potentially Mist endocytosis is downregulated in the mesoderm compared to the ectoderm. Interestingly, the E3 ubiquitin ligase Neuralized (Neur), which is uniformly expressed in the embryo, is inhibited in the ectoderm by the small proteins of the Bearded (Brd) family. Brd genes are repressed by the mesoderm transcription factor Snail, so that Neur is only active in the mesoderm. In a Brd mutant, where Neur becomes active in the ectoderm, MyoII activation is increased and Neur degradation or repression in the mesoderm following Brd overexpression both reduce MyoII activation. Previous studies have shown that the E3 ubiquitin ligase targets β-arrestin-2 for ubiquitination and degradation, and, thereby, it affects endocytosis and signaling by GPCRs. It is possible that GPCR endocytosis could be reduced in the mesoderm due to increased Neur activity in this tissue. This may depend on the downregulation of several target proteins, such as β-arrestin-2 (Jha, 2018).

Selective compartmentalization of GPCR on the plasma membrane as in the case of large apical plasma membrane invaginations can increase the concentration and the probability of GPCR clustering and oligomerization. The current data suggest that the dynamic modulation of GPCR signaling can be achieved by a change in their cluster/oligomer formation. Receptor oligomerization may enlarge the signaling capacities by the recruitment of more downstream signaling components during GPCR signaling. G proteins are reported to be expressed at low concentration, and selective compartmentalization of GPCRs on the plasma membrane further increase the probability of GPCR clustering and oligomerization for efficient signaling. Investigation of G protein activation by different GPCRs in vivo will be needed to test if a similar mechanism is in place during epithelial morphogenesis (Jha, 2018).

Homodimerization of Drosophila Class A neuropeptide GPCRs: Evidence for conservation of GPCR dimerization throughout metazoan evolution

While many instances of GPCR dimerization have been reported for vertebrate receptors, invertebrate GPCR dimerization remains poorly investigated, with few invertebrate GPCRs having been shown to assemble as dimers. To date, no Drosophila GPCRs have been shown to assemble as dimers. To explore the evolutionary conservation of GPCR dimerization, an acceptor-photobleaching FRET methodology was used to evaluate whether multiple subclasses of Drosophila GPCRs assembled as homodimers when heterologously expressed in HEK-293 T cells. Multiple Drosophila neuropeptide GPCRs that exhibited structural homology with a vertebrate GPCR family member previously shown to assemble as a dimer were C-terminally tagged with CFP and YFP fluorophores, and these receptors were visualized through confocal microscopy. FRET responses were determined based on the increase in CFP emission intensity following YFP photobleaching for each receptor pair tested. A significant FRET response was observed for each receptor expressed as a homodimer pair, while non-significant FRET responses were displayed by both cytosolic CFP and YFP expressed alone, and a heterodimeric pair of receptors from unrelated families. These findings suggest that receptors exhibiting positive FRET responses assemble as homodimers at the plasma membrane and are the first to suggest that Drosophila GPCRs assemble as homodimeric complexes. It is proposed that GPCR dimerization arose early in metazoan evolution and likely plays an important and underappreciated role in the cellular signaling of all animals (Rizzo, 2019).

G-protein signaling is required for increasing germline stem cell division frequency in response to mating in Drosophila males

Adult stem cells divide to renew the stem cell pool and replenish specialized cells that are lost due to death or usage. However, little is known about the mechanisms regulating how stem cells adjust to a demand for specialized cells. A failure of the stem cells to respond to this demand can have serious consequences, such as tissue loss, or prolonged recovery post injury. This study challenged the male germline stem cells (GSCs) of Drosophila melanogaster for the production of specialized cells, sperm cells, using mating experiments. Repeated mating reduced the sperm pool and increased the percentage of GSCs in M- and S-phase of the cell cycle. The increase in dividing GSCs depended on the activity of the highly conserved G-proteins. Germline expression of RNA-Interference (RNA-i) constructs against G-proteins, or a dominant negative G-protein eliminated the increase in GSC division frequency in mated males. Consistent with a role for the G-proteins in regulating GSC division frequency, RNA-i against seven out of 35 G-protein coupled receptors (GPCRs) within the germline cells also eliminated the capability of males to increase the numbers of dividing GSCs in response to mating (Malpe, 2020).

This study shows that repeated mating reduced the sperm pool and increased GSC division frequency. Using highly controlled experiments, it was demonstrated that mated males had more GSCs in M-phase and S-phase of the cell cycle compared to non-mated males. Mated males also showed faster incorporation of EdU in feeding experiments, suggesting that the GSCs of mated males entered the S-phase of the cell cycle more frequently. Though the possibility that mated males ingested more EdU-supplemented food than their non-mated siblings cannot be excluded, the data suggest that the GSCs in mated males cycle faster. The response curve obtained in a time-course experiment is different from the response curves reported by other groups that used bromo-deoxy-uridine (BrDU) as the thymidine analog instead of EdU. For example, the non-mated males in the current experiment had about 70% of EdU-positive GSCs after 48 hours of feeding. A study that uses white (w) mutant animals and fed the same concentration of the thymidine homologue had a steeper response curve, in which 85% of the GSCs were BrDU-marked after 48 hours of feeding. Another study using y, v flies showed even steeper response curves where 100% of the GSCs were BrDU-labeled after 24 hours. However, in this study, animals were fed a 30 times higher concentration of the thymidine homologue than used in the current study. It is proposed that the different response curves are due to the different genetic backgrounds, chemicals, and doses (Malpe, 2020).

These findings demonstrate that GSCs can respond to a demand for sperm by increasing their mitotic activity. Based on RNA-i targeting G-proteins and a dominant negative construct against Gγ1, the increase in MIGSC of mated males is dependent on G-protein signaling. Furthermore, signal transducers predicted to act downstream of G-proteins and GPCRs predicted to act upstream of G-proteins also appeared to be required for the response to mating. Whether G-protein signaling directly affects GSC division frequency, or whether G-protein-dependent communication among the early stage germline cells impacts the MIGSC remains to be investigated (Malpe, 2020).

Due to the lack of mutants and a potential interference of whole animal knock-down in the behavior of the flies, tissue-specific expression of RNA-i-constructs was used. It is surprising that these studies revealed potential roles for seven instead of a single GPCR in the increase of MIGSC in response to mating. A possible explanation is that some of the RNA-i-lines have off-target effects. RNA-i-hairpins can cause the down-regulation of unintended targets due to stretches of sequence homologies, especially when long hairpins are used. However, with the exception of the RNA-i-line directed against 5-HT7, all lines that produced a phenotype contain second generation vectors with a short, 21 nucleotide hairpin predicted to have no off-target effects. Thus, it is hypothesized that multiple GPCRs regulate the increase in MIGSC in response to mating. Consistent with this, expression of other RNA-i-lines directed against Mth or 5-HT1A interfered with the increase in MIGSC in mated males (Malpe, 2020).

The finding that RNA-i against several GPCRs blocked the increase in MIGSC in mated males suggests a high level of complexity in the regulation of GSC division frequency. One simple explanation could be that the increase in GSC division frequency is dependent on ideal physiological conditions and that lack of any of the seven GPCRs somehow impairs the cell's normal metabolism. Alternatively, the GPCRs could act in concert to impact the MIGSC. In the literature, increasing evidence has emerged that GPCRs can form dimers and oligomers and that these have a variety of functional roles, ranging from GPCR trafficking to modification of G-protein mediated signaling. In C. elegans, two Octopamine receptors, SER-3 and SER-6, additively regulate the same signal transducers for food-deprived-mediated signaling. One possible explanation for the non-redundant function of the two receptors was the idea that they form a functional dimer. In mammalian cells, 5-HT receptors can form homo-dimers and hetero-dimers and, dependent on this, have different effects on G-protein signaling. In cultured fibroblast cells, for example, G-protein coupling is more efficient when both receptors within a 5-HT4 homo-dimer bind to agonist instead of only one. In cultured hippocampal neurons, hetero-dimerization of 5-HT1A with 5-HT7 reduces G-protein activation and decreases the opening of a potassium channel compared to 5-HT1A homo-dimers59. The formation of hetero-dimers of GPCRs with other types of receptors plays a role in depression and in the response to hallucinogens in rodents (Malpe, 2020).

Alternatively, or in addition to the possibility that some or all of the seven GPCRs form physical complexes, a role for several distinct GPCRs in regulating GSC division frequency could be explained by cross-talk among the downstream signaling cascades. One signaling cascade could, for example, lead to the expression of a kinase that is activated by another cascade. Similarly, one signaling cascade could open an ion channel necessary for the activity of a protein within another cascade. Unfortunately, the literature provides little information on Drosophila GPCR signal transduction cascades and only very few mutants have been identified that affect a process downstream of GPCR stimulation. Thus, it remains to be explored how stimulation of the GPCRs and G-proteins increase GSC divisions (Malpe, 2020).

The role for G-protein signaling in regulating the frequency of GSC divisions is novel. The data suggest that the increase in MIGSC in response to mating is regulated by external signals, potentially arising from the nervous system, that stimulate G-protein signaling in the germline. Based on the nature of the GPCRs, the activating signal could be Serotonin, the Mth ligand, Stunted, Octopamine, or two other, yet unknown, signals that activate Mth-l5, and CG12290. It will be interesting to address which of these ligands are sufficient to increase MIGSC, in what concentrations they act, by which tissues they are released, and whether they also affect other stem cell populations (Malpe, 2020).

Proteolytic activation of Growth-blocking peptides triggers calcium responses through the GPCR Mthl10 during epithelial wound detection

The presence of a wound triggers surrounding cells to initiate repair mechanisms, but it is not clear how cells initially detect wounds. In epithelial cells, the earliest known wound response, occurring within seconds, is a dramatic increase in cytosolic calcium. This study shows that wounds in the Drosophila notum trigger cytoplasmic calcium increase by activating extracellular cytokines, Growth-blocking peptides (Gbps), which initiate signaling in surrounding epithelial cells through the G-protein-coupled receptor Methuselah-like 10 (Mthl10). Latent Gbps are present in unwounded tissue and are activated by proteolytic cleavage. Using wing discs, this study showed that multiple protease families can activate Gbps, suggesting that they act as a generalized protease-detector system. Experimental and computational evidence is presented that proteases released during wound-induced cell damage and lysis serve as the instructive signal: these proteases liberate Gbp ligands, which bind to Mthl10 receptors on surrounding epithelial cells, and activate downstream release of calcium (O'Connor, 2021).

GPCR voltage dependence controls neuronal plasticity and behavior

The G-protein coupled receptors (GPCRs) play a paramount role in diverse brain functions. Almost 20 years ago, GPCR activity was shown to be regulated by membrane potential in vitro, but whether the voltage dependence of GPCRs contributes to neuronal coding and behavioral output under physiological conditions in vivo has never been demonstrated. This study shows that muscarinic GPCR mediated neuronal potentiation in vivo is voltage dependent. This voltage dependent potentiation is abolished in mutant animals expressing a voltage independent receptor. Depolarization alone, without a muscarinic agonist, results in a nicotinic ionotropic receptor potentiation that is mediated by muscarinic receptor voltage dependency. Finally, muscarinic receptor voltage independence causes a strong behavioral effect of increased odor habituation. Together, this study identifies a physiological role for the voltage dependency of GPCRs by demonstrating crucial involvement of GPCR voltage dependence in neuronal plasticity and behavior. Thus, this study suggests that GPCR voltage dependency plays a role in many diverse neuronal functions including learning and memory (Rozenfeld, 2021).

Currently, the only known voltage sensor of GPCRs is for muscarinic receptors. The Drosophila olfactory system is cholinergic and has high expression levels of muscarinic receptors. Only two Drosophila muscarinic receptors are expressed in the fly brain: a Gq coupled Drosophila muscarinic type A receptor (mAChR-A, homologous to M1R); and a Gi/o coupled Drosophila muscarinic type B receptor (mAChR-B, homologous to M2R). Although the Drosophila genome contains a third Drosophila muscarinic type C receptor, its expression level in the brain is negligible. As a result, manipulation of Drosophila muscarinic receptors results in profound physiological and behavioral effects, which makes the Drosophila olfactory system well suited to examine whether GPCR voltage dependence affects physiological processes and behavior (Rozenfeld, 2021).

Odors activate Drosophila cholinergic olfactory receptor neurons (ORNs) that are located in the antennae and maxillary palps. Each ORN expresses a single odorant receptor gene. ORNs expressing the same receptor send their axons to a single glomerulus in the antennal lobe (AL, homologous to the mammalian olfactory bulb). Second-order excitatory cholinergic projection neurons (ePNs) send their dendrites to a single glomerulus and serve as the primary output channel of the AL. The AL also contains multi glomeruli inhibitory GABAergic and glutamatergic local neurons (iLNs) that receive input from ORNs and PNs (Rozenfeld, 2021).

It was recently demonstrated that mAChR-A is mainly localized to a subpopulation of GABAergic iLNs where it induces short-term potentiation of iLNs. Knockdown of mAChR-A in iLNs also causes changes of odor valence. In addition to odor valence, iLN activity is required for odor habituation, sustained odor responses, regulating gain control, signal separation, and enhancement of interglomerular contrast (Rozenfeld, 2021).

This study shows that the Drosophila mAChR-A, which diverged from the mammalian muscarinic receptors over 700 million years ago is also voltage dependent. CRISPR/Cas9 was used to generate a fly strain with two point mutations that abolish the mAChR-A voltage dependence while retaining normal maximal activity. This enables demonstration that wt mAChR-A induced post-tetanic potentiation (PTP) is voltage dependent and that this dependency is abolished in mutant flies expressing a voltage independent mAChR-A. Even more striking is that depolarization alone, without any agonist, causes a potentiation of nicotinic receptors in iLNs which is mediated by mAChR-A. In addition, the generation of a voltage-independent mAChR-A results in a pronounced behavioral effect of increased odor habituation, which was localized to iLNs. Taken together, this study provides a demonstration of a physiological role for the voltage dependency of GPCRs, and may serve as a paradigm shift in understanding of neural function and drug discovery (Rozenfeld, 2021).

This study shows that the Drosophila mAChR-A is voltage dependent. In addition, the results reveal that a voltage-independent receptor variant, mAChR-A-KK, exhibits altered neuronal potentiation to both artificial and physiological stimuli, and that these changes in potentiation influence behavior. Furthermore, generating conditions such that mAChR-A becomes voltage independent and resides in the low activity state, completely abolishes the mAChR-A dependent potentiation. Thus, this work represents a demonstration that voltage dependence is crucial for the normal function of GPCRs in vivo, thereby changing understanding of GPCR recruitment and function (Rozenfeld, 2021).

GPCRs voltage dependence was discovered almost 20 years ago. Since then it was demonstrated for various GPCRs. These studies provided important information on the identity of voltage-dependent GPCRs as well as on some of the mechanisms underlying GPCR voltage dependency. However, as they were performed in cell culture, whether this GPCR voltage dependency plays any physiological role was not addressed. GPRC voltage dependence was shown to control synaptic release initiation and duration in vitro, and recently it was shown that membrane depolarization recruits voltage-dependent purinergic receptors in sympathetic chromaffin cells to increase the quantal size. Nevertheless, even in these cases, there is no evidence that these small changes in the duration or strength of synaptic release affect neuronal computation or behavioral output, especially on the background of noisy neural activity. This study, which generated a fly strain with a voltage independent muscarinic receptor allowed this question to be addressed, and it was unequivocally demonstrated that GPCR voltage dependence affects neuronal computation and behavioral output. Furthermore, contrary to all previous studies which showed effects only on synaptic release, this study demonstrates that GPCR voltage dependence plays a role postsynaptically in the canonical GPCR pathway (Rozenfeld, 2021).

Since mAChR-A voltage dependence shifts the dynamic range of the dose-response curve, it can only be relevant if mAChR-A is exposed to sub-saturation concentrations of neurotransmitter. In this context, mammalian glutamatergic receptors are usually far from saturation during quantal transmission. Notably, the rapid removal of neurotransmitter from the synaptic cleft also generates sub-saturation conditions. The current results further support this notion since mAChR-A-KK has similar activity to mAChR-A at saturation levels of the receptor, and differs only in the dynamic range of responses. The findings of a strong physiological effect in response to synaptic release as well as a strong behavioral effect all point to a sub-saturating agonist concentration (Rozenfeld, 2021).

The demonstration that GPCR voltage dependence has physiological implications, suggests the presence of a strong crosstalk between the ionotropic pathways (that rapidly affect membrane potential) and the metabotropic pathways. For GPCRs that exhibit increased activity upon depolarization, this crosstalk is reminiscent of the crosstalk between the glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR). NMDAR activity requires depolarization, which usually originates from AMPAR activation, and the recruitment of NMDAR can result in AMPA potentiation. Although the metabotropic receptors do not require depolarization for their activity, depolarization favors their recruitment. For nicotinic and muscarinic receptors, a model arises where depolarization caused by activation of nicotinic receptors strongly affects the activity of the co-activated muscarinic receptor. In turn, the improved recruitment of the muscarinic receptors results in potentiation of the nicotinic receptors. The above suggestion is not limited only to the muscarinic receptors but can also be extended to other GPCRs. For example, a strong similarity was recently demonstrated between the function of mAChR-A in iLNs and the function of the glutamatergic mGluR1 expressed in inhibitory granule cells of the mammalian olfactory bulb. It is interesting to note that this similarity is further extended to their voltage dependence and mGluR1 shifts to a high activity state following depolarization. It is thus possible that similar to the Drosophila muscarinic receptor, mGluR1 voltage dependency participates in a crosstalk between glutamatergic ionotropic and metabotropic receptors and has a role in mammalian olfactory processing (Rozenfeld, 2021).

In Drosophila, odor habituation is thought to be mediated by the potentiation of iLNs. The current results are in agreement with the role of iLNs in odor habituation and demonstrate that cholinergic neuromodulation by mAChR-A is an important step in odor habituation. The results further show that mAChR-A voltage dependence plays a role in odor habituation. It is interesting to note that odor habituation requires PN input onto iLNs54. The requirement of PN input can explain how odor habituation occurs in a glomerulus-specific manner (i.e. according to the identity of the activated PNs). However, PNs generally respond in a non-linear manner to different concentrations, with relatively low odor concentrations required to saturate PN activity. Thus, even low odor concentrations should generate strong odor habituation, contrary to observed results. The results, which show that mAChR-A voltage dependence plays a role in odor habituation, may resolve this discrepancy. Contrary to PNs, iLNs are linearly recruited by ORNs. Thus at low odor concentration iLNs will undergo only weak depolarization. Therefore, although the relevant PN will already be saturated, due to the low iLN depolarization, the responding mAChR-A will be in the low activity state and the overall habituation will not be strong. In contrast, at a high odor concentration, iLNs will undergo strong depolarization, shifting mAChR-A to the high activity state, and as a result habituation will be stronger. In this way, GPCR voltage dependence can act as a rheostat that allows for a gradual increase in neuromodulation (Rozenfeld, 2021).

Taken together, this work provides a demonstration of a physiological role for the voltage dependency of GPCRs and may serve as a paradigm shift in understanding of neural function and drug discovery (Rozenfeld, 2021).

The incidence of candidate binding sites for β-arrestin in Drosophila neuropeptide GPCRs

To support studies of neuropeptide neuromodulation, beta-arrestin binding sites (BBS's) by evaluating the incidence of BBS sequences among the C terminal tails (CTs) of each of the 49 Drosophila melanogaster neuropeptide GPCRs. BBS were identified by matches with a prediction derived from structural analysis of rhodopsin:arrestin and vasopressin receptor: arrestin complexes. To increase the rigor of the identification, the conservation was determined of BBS sequences between two long-diverged species D. melanogaster and D. virilis. There is great diversity in the profile of BBS's in this group of GPCRs. Evidence is presented for conserved BBS's in a majority of the Drosophila neuropeptide GPCRs; notably some have no conserved BBS sequences. In addition, certain GPCRs display numerous conserved compound BBS's, and many GPCRs display BBS-like sequences in their intracellular loop (ICL) domains as well. Finally, 20 of the neuropeptide GPCRs are expressed as protein isoforms that vary in their CT domains. BBS profiles are typically different across related isoforms suggesting a need to diversify and regulate the extent and nature of GPCR:arrestin interactions. This work provides the initial basis to initiate future in vivo, genetic analyses in Drosophila to evaluate the roles of arrestins in neuropeptide GPCR desensitization, trafficking and signaling (Taghert, 2022).

Local 5-HT signaling bi-directionally regulates the coincidence time window for associative learning

The coincidence between conditioned stimulus (CS) and unconditioned stimulus (US) is essential for associative learning; however, the mechanism regulating the duration of this temporal window remains unclear. This study found that serotonin (5-HT) bi-directionally regulates the coincidence time window of olfactory learning in Drosophila and affects synaptic plasticity of Kenyon cells (KCs) in the mushroom body (MB). Utilizing GPCR-activation-based (GRAB) neurotransmitter sensors, this study found that Kenyon cell (KC)-released acetylcholine (ACh) activates a serotonergic dorsal paired medial (DPM) neuron, which in turn provides inhibitory feedback to KCs. Physiological stimuli induce spatially heterogeneous 5-HT signals, which proportionally gate the intrinsic coincidence time windows of different MB compartments. Artificially reducing or increasing the DPM neuron-released 5-HT shortens or prolongs the coincidence window, respectively. In a sequential trace conditioning paradigm, this serotonergic neuromodulation helps to bridge the CS-US temporal gap. Altogether, this study reports a model circuitry for perceiving the temporal coincidence and determining the causal relationship between environmental events (Zeng, 2023).

Involvement of neuronal tachykinin-like receptor at 86C in Drosophila disc repair via regulation of kynurenine metabolism

Neurons contribute to the regeneration of projected tissues; however, it remains unclear whether they are involved in the non-innervated tissue regeneration. This study shows that a neuronal tachykinin-like receptor at 86C (TkR86C) is required for the repair of non-innervated wing discs in Drosophila. Using a genetic tissue repair system in Drosophila larvae, genetic screening was performed for G protein-coupled receptors to search for signal mediatory systems for remote tissue repair. An evolutionarily conserved neuroinflammatory receptor, TkR86C, was identified as the candidate receptor. Neuron-specific knockdown of TkR86C impaired disc repair without affecting normal development. The humoral metabolites of the kynurenine (Kyn) pathway regulated in the fat body were investigated because of their role as tissue repair-mediating factors. Neuronal knockdown of TkR86C hampered injury-dependent changes in the expression of vermillion in the fat body and humoral Kyn metabolites. These data indicate the involvement of TkR86C neurons upstream of Kyn metabolism in non-autonomous tissue regeneration (Kashio, 2023).

The adhesion G-protein-coupled receptor mayo/CG11318 controls midgut development in Drosophila

Adhesion G-protein-coupled receptors (aGPCRs) form a large family of cell surface molecules with versatile tasks in organ development. Many aGPCRs still await their functional and pharmacological deorphanization. This study characterized the orphan aGPCR mayo^KO (identity crisis) mutants can be restored by pharmacological inhibition of potassium channels. Intriguingly, hyperkalemia and tachycardia are caused non-cell autonomously through mayo-dependent control of enterocyte proliferation in the larval midgut, which is the primary function of this aGPCR. These findings characterize the ancestral aGPCR Mayo as a homeostatic regulator of gut development (Contreras, 2024).

The gastrointestinal (GI) tracts in Drosophila and vertebrate species share features of anatomical and functional compartmentalization. The foregut (equivalent to the mammalian esophagus) passes ingested food to the crop (stomach), the anterior midgut (small intestine) controls nutrient, ion, and water absorption, and, finally, the hindgut (large intestine) is involved in electrolyte reabsorption from the Malpighian tubules (equivalent to the kidney in mammals). Instead of the crypt-villus structure found in mammals, the Drosophila midgut consists of a monolayer populated by different cell types. Before pupation, the larval midgut is composed of enterocytes (ECs), which derive from principal midgut epithelial cells (PMECs), interstitial cell precursors (ICPs), and adult midgut precursors (AMPs), which generate and are enveloped by peripheral cells (PCs). AMPs are also thought to give rise to enteroendocrine cells (EEs). The larval AMPs, ensheathed by PCs, constitute a stem cell niche for transient pupal midgut development until metamorphosis and also the generation of the adult midgut. Dysregulation of midgut cell proliferation can lead to malabsorption of nutrients and defective ion homeostasis (Contreras, 2024).

Adhesion G-protein-coupled receptors (aGPCRs) are a large group of surface sensors with various functions in tissue development. For example, aGPCRs exert control of vital aspects of embryogenesis and cardiogenesis, as well as nervous and immune systems development. Genetic dysfunctions of aGPCRs are associated with various human pathologies such as multiple cancers, neurodevelopmental disord ol over asymmetric mitotic activity of PMECs to yield a fixed EC pool. As previously shown, the aGPCR Cirl exerts a similar effect to adjust the number of neuroblasts in the larval brain of Drosophila melanogaster, and its homolog LAT-1 controls mitotic spindle positioning and division planes of early blastomeres in C. elegans (Contreras, 2024).

Also, adult guts of mayo^KO flies were elongated. A similar phenotype was reported after overexpression of CD97/ADGRE5 in mouse intestinal epithelial cells, which caused a mega-intestine through cylindrical growth, showing that aGPCRs partake in GI development in other species too (Contreras, 2024).

In order to clarify which cell type relies on mayo function, this study harnessed the observation that mayo^KO larvae display a marked non-cell-autonomous tachycardia likely caused by a mild hyperkalemia. This is in line with the role of K+ currents in the repolarization of the Drosophila heart, while Na+ does not affect the heart frequency in Drosophila. Further, cardiac action potentials in invertebrates are carried by K+ and Ca2+ fluxes, and K+ channel mutations can cause cardiac arrhythmias, confirming the involvement of K+ channels in the regulation of the cardiac pacemaker (Contreras, 2024).

This study utilized the tachycardia in mayo^KO larvae in a screen to test which cells require mayo autonomously to maintain physiological K+ hemolymph levels. Only removal of mayo from PCs and ECs resulted in tachycardiac frequencies, showing that mayo is not only expressed but also required in those cells for the maintenance of a normal cardiac heart frequency (Contreras, 2024).

The first characterization of the aGPCR mayo in Drosophila has provided insights into its role in the larval midgut as a potential mitotic suppressor at the PMEC-EC transition. Future studies will need to clarify which molecular function mayo serves in ECs and PCs and how it does so. To this end, it is necessary to determine Mayo ligands and intracellular signaling conduits to reconstruct the receptor's signaling pathway. In particular, how Mayo is integrated into the mechanisms of EC proliferation will be of prime interest. This will shed light on how removal of mayo causes an increase in EC number and whether its mode of action is comparable to effects of other aGPCRs on mitotic activity, cell polarity, division, and fate determination (Contreras, 2024).

In conjunction with the proliferative effect of mayo removal on ECs it is concluded that this cell type likely affects K+ uptake into the hemolymph either directly through ion channels or transporters/exchange proteins, or indirectly by controlling the abundance of enteric cell types that express them. Thus, future work needs to address whether the extracellular potassium concentration simply scales with the EC number, or whether mayo impacts potassium flux phenomena in ECs as well (Contreras, 2024).

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Zygotically transcribed genes

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