BIOLOGICAL OVERVIEW

Pollux (Plx) is a cell adhesion protein expressed in the CNS and trachea, and oocytes. Loss of Plx function results in a progressive accumulation of fluid within the larva's tracheal network, culminating in congestion throughout the trachea and ultimately death due to asphyxiation. During embryonic development, Plx is deposited uniformly on the apical surface of cellular blastoderm cells, and is later regionalized along subsets of CNS axon pathways and on the lumen surface of large diameter tracheal tubes. While cell-attachment studies show that Plx can serve as a ligand for Arg-Gly-Asp (RGD) binding integrins, Plx also contains additional extracellular matrix (ECM) motifs and cell-adhesion motifs, as well as a leucine zipper. Plx's unique structural features exclude its placement in any of the previously described cell adhesion molecules (CAMs) or ECM gene families. However, data-base searches have identified proteins in plants, yeasts, nematodes, and humans with similar structures but whose cellular functions are currently unknown. In addition to a novel 74aa domain, these proteins also share putative ECM/cell-adhesion motifs and potential membrane anchoring domains. This suggests that, like Plx, they too may function as membrane associated adhesion molecules (Zhang, 1996).

The expression pattern of Pollux in the CNS is reminiscent of Roundabout (Robo). Starting at stage 13, Plx begins to accumulate uniformly along axon tracts that extend the entire length of the ventral cord's longitudinal connectives. However, no appreciable Plx immunostaining is observed in the ventral cord's commissures. Similarly, little or no Robo expression is observed on commissural growth cones as they extend toward and across the midline. However, as these growth cones turn to project longitudinally, their levels of Robo expression dramatically increase. Robo is expressed at high levels on all longitudinally projecting growth cones and axons. In contrast to this, Robo is expressed at nearly undetectable levels on commissural axons (Kidd, 1998). pollux mutation gives no CNS phenotype, suggesting that maternal contribution is sufficient for CNS Plx function or that backup systems exist in the CNS to overcome Plx deficiency (Zhang, 1996). Nevertheless, to date there has been no analysis of a possible interaction between robo and plx.

What is the basis for the tracheal defect in larvae leading to mortality in plx mutants? To determine if structural defects exist in the larval trachea of plx mutants, which would be unresolvable by light microscopy, larval ultrastructure was examined. Electron microscopic views of serial transverse sections, collected from the seventh and eighth abdominal segments of 6-10 hr old and older larva, fail to identify structural defects within the trachea's epithelial cells or in their contacts with other epithelial cells. No breaks between cells are observed and their interlocking tight junctions appear normal. In addition, the outer basement lamina of plx- trachea, surrounding the tracheal epithelium's outer surface, also appear normal. However, structural differences between wild-type and plx- tracheae are observed in the epithelial cell's cuticular ECM. In wild-type trachea, the pre-molt cuticle consists of a regularly folded (electron-dense) epicuticle that protrudes into and lines the lumen cavity. Underlying the epicuticle is an inner, more electron-lucent, endo- or pro-cuticle. Taenidia are spiral sclerotized fibers that stiffen the walls of the tracheae of insects. The pro-cuticle, found in larger diameter tubes, consists of a fibrillar matrix that fills the intrataenidial area between the epicuticle and the surface of the epithelial cells. In smaller branches of the trachea, the procuticle becomes thinner and is absent from the tracheoles. Although the apical surface of the cuticle appear normal and no structural breaks are detected, the epicuticle of the plx- trachea is separated from the underlying epithelium. In addition, the procuticle's fibrous matrix is also reduced or missing in the taenidial folds. The loss of epicuticle contact with the epithelium and the reduced/disorganized filamentous network within the procuticle is particularly evident in the major, large and intermediate tracheae. Conversely, no significant differences between wild-type and plx- are observed in the small diameter tubes. The loss of the fibrillar matrix within the procuticle appears greatest in the taenidial folds where clearings devoid of fibrous material are frequently observed. The cuticular ECM defects are observed in plx- tracheae with and without detectable levels of excessive fluid accumulation (Zhang, 1996).

The tracheal epithelium's reduced contact with its epicuticle and the observed disorganization in the procuticle's fibrous matrix of plx- trachea suggests that Plx may function to ensure proper epithelium-epicuticle contact via adhesive links with the ECM. When released from the epithelial surface, Plx may also provide an organizational role in the procuticle's filamentous-matrix by cross-linking components of the procuticle's ECM. During apolysis, a process whereby the tracheal epithelium sheds its cuticle, ECM degrading enzymes are released by the epithelium, triggering the epicuticle's separation from the epithelium. Premature apolysis could be induced by a number of factors, including loss of ECM contact and/or a premature exogenous signal to initiate apolysis. At the moment, these two possibilities cannot be distinguished. However, in the absence of any observable breaks in the plx- epicuticle, an event associated with apolysis, these findings suggest that the epithelial-ECM separation is due to loss of adhesive contacts and/or defects in the procuticle's organization. The reduced contact most likely compromises the trachea's structural integrity, interfering with its ability to insulate/seal-off its lumen from body fluids (Zhang, 1996).

To identify Calmodulin-binding proteins that may function in phototransduction and/or synaptic transmission, a screen was conducted for retinal Calmodulin-binding proteins. Twelve Calmodulin-binding proteins were found that are expressed in the Drosophila retina. The functions of Calmodulin appear to be mediated, at least in part, by four previously identified calmodulin-binding proteins: the Trp and Trp-like ion channels, NinaC and InaD. Eight calmodulin-binding proteins have been identified that have not been previously reported to be expressed in the Drosophila retina. The full-length sequences corresponding to three of the calmodulin-binding proteins have been described. These corresponded to two Calmodulin-dependent protein kinases, MLCK and CaM kinase II, as well as to one of two previously described Calcineurin proteins. A third calmodulin-dependent protein kinase is expressed in the Drosophila retina, CaM kinase I. The remaining four calmodulin-binding proteins have not been known to bind calmodulin prior to the current work. Six targets have been found that are related to proteins implicated in synaptic transmission. Among these six are a homolog of the diacylglycerol-binding protein (UNC13) and a protein (CRAG) related to Rab3 GTPase exchange proteins. Two other calmodulin-binding proteins found are Pollux, a protein with similarity to a portion of a yeast Rab GTPase activating protein, and Calossin, an enormous protein of unknown function conserved throughout animal phylogeny. Thus, it appears that Calmodulin functions as a Ca2+ sensor for a broad diversity of retinal proteins, some of which are implicated in synaptic transmission (Xu, 1998).

A possible clue as to the function of Plx in the retina is that it shares some similarity to two yeast Rab GAP proteins, although no homology was found to the Rab3 GAP expressed in the rat brain. Nevertheless, the observation that Plx contains a domain related to Rab GAPs combined with the finding that it appears to be localized to the plasma membrane and lumen of the trachael system raises the possibility that Plx may be involved in exocytosis. In the Drosophila visual system, exocytosis is important not only in synaptic transmission but in turn-over of the microvillar membrane of the photoreceptor cells. Shedding of membrane does not occur uniformly during the diurnal cycle, but occurs maximally soon after dawn. Thus, an increase in the exocytotic process is correlated with the light dependent rise in Ca²⁺ and therefore might be regulated in part by a Ca²⁺ sensing component in a Rab cycle. Alternative potential functions for Plx in photoreceptor cells include other processes that involve vesicular trafficking such as insertion of new membrane in the microvilli and the budding, targeting, and fusion of rhodopsin carrier vesicles with the plasma membrane. These latter events involve a variety of Rab proteins and also appear to be regulated during the daily light cycle (Xu, 1998 and references).

GENE STRUCTURE

castor and pollux are separated by 99bp and transcribed from opposite strands. Deduced from DNA sequence analysis of overlapping cDNA and genomic clones, the transcribed sequence of plx consists of four exons separated by three small AT rich introns that are 58, 55 and 68 bp in length. Primer extension and RNase protection experiments, using embryo mRNA, indicate that plx transcription initiates 22 bp 5' to the longest isolated cDNA (Zhang, 1996).

Exons - 4

PROTEIN STRUCTURE

Amino Acids - 732 (Zhang, 1996) and 1379 (Xu, 1998)

Structural Domains and Evolutionary Homologs

Pollux (Plx) is a protein previously reported to be 732 amino acids in length and required for viability (Zhang, 1996). The protein is predicted to have a transmembrane domain and a leucine zipper (Zhang, 1996). Plx has now been found to be 1379 amino acids in length and the formerly assigned initiator methionine corresponds to residue 648. A protein related to Plx is TBC1 (Richardson, 1995), a mouse protein which had homology to the majority of Plx. The region in Plx that contains the greatest similarity to TBC1 is a 337-amino acid segment (51% identity, residues 676-1012) that includes the putative transmembrane domain. Of particular interest, the region most highly conserved between Plx and TBC1 includes a 153-amino acid domain (residues 811-963) that displays moderate homology to the yeast Rab family GTPase-activating proteins, GYP6 or GYP7 (Strom, 1994). GYP7 is ~29% identical to this domain in either Plx or TBC1; however, if two gaps of 18 and 36 amino acids have been introduced in Plx and TBC1, the 29% homology extends to over 222 amino acids (742-963). This ~200 amino acid sequence corresponds to the domain previously referred to as a TBC domain due to its similarity to segments in the TRE-2 oncogene and the yeast regulators of mitosis, BUB2 and CDC16. TBC1 is 1141 residues and is found to be a nuclear protein. Thus, TBC1 and Plx have very disparate spatial distributions. (Xu, 1998).

The portion of the Plx protein that was isolated in the screen extends from residues 180-1379. Using a series of overlapping GST fusion proteins and the gel overlay assay, the calmodulin-binding site(s) contained in the original fusion protein was further mapped to residues 657-680. The sequence of the calmodulin-binding site is not conserved in the mouse homolog, TBC1, but is in several human ESTs. A bovine homolog of Plx (Lyncein), which was isolated from a bovine retinal library, is highly conserved in the calmodulin-binding domain despite having no higher overall sequence conservation to Plx than TBC1. Moreover, a fusion protein containing the conserved sequence in Lyncein binds calmodulin. Plx also bind to calmodulin in a pull-down assay; although this interaction is Ca²⁺ independent (Xu, 1998).

Thus it has been found that Plx is 1379 residues rather than 732 amino acids as previously reported (Zhang, 1996). The additional sequence is not due to a chimeric cDNA since multiple plx cDNAs were obtained and TBC1 shares similarity to Plx both N- and C-terminal to the formerly assigned initiating methionine at residue 648. Plx has been shown to bind calmodulin and does so in a Ca²⁺-independent manner. Although the sequence of the calmodulin-binding site is not conserved in TBC1, the region is very similar in Lyncein, a homolog isolated from a bovine retinal library. Furthermore, the Lyncein sequence also binds calmodulin. Thus, it appears that a Plx homolog is expressed in the vertebrate retina (Xu, 1998).

Marked by a novel 74-amino-acid domain, Plx belongs to a highly conserved family with members in plants, yeasts, nematodes, and humans, including the human oncoprotein TRE17. Plx also contains a motor neuron-selective adhesive site, multiple proteoglycan-binding motifs, and a leucine zipper: all suggest possible associations with additional components of the adhesion complex. Plx has an RGD integrin recognition/attachment site [a Leu-Arg-Glu (LRE) motor neuron-selective attachment motif first identified in vertebrate S-laminin (Hunter, 1989)], multiple proteoglycan-adhesion sites and potential disulfide bridging residues. Within its presumptive intracellular domain, Plx also contains a protein-protein dimerization domain, the alpha helical leucine zipper. Secondary structure predictions indicate the 29aa residues spanning positions 411-439 have a high probability of forming an amphipathic alpha helix, which satisfies the leucine zipper's 4-3 hydrophobic rule. Leucine zippers have been found in other CAM and ECM glycoproteins, such as the Drosophila laminin B1 chain, suggesting that in addition to potentially forming homodimers, Plx may dimerize with other proteins. A hydropathicity plot, employed to predict hydrophilic and membrane-associated hydrophobic domains, indicates that a centrally located stretch of 44 predominantly non-polar amino acids (residues 262-306) has a sufficient number of hydrophobic residues to span a lipid bilayer twice. The hydropathicity plot also reveals that Plx lacks an obvious amino-terminal transmembrane insertion signal peptide. The absence of an N-terminal signal sequence in transmembrane or secreted proteins is not unprecedented. The transmembrane proteins p33, band 3 and the influenza neuraminidase all lack N-terminal signal sequences; moreover, the secreted glycoprotein ovalbumin has been shown to contain an internal signal sequence. Although the precise membrane polarity of Plx is currently unknown, its central hydrophobic domain may serve a dual function both as a transmembrane insertion signal and as a membrane anchor. An additional hydrophobic region spanning residues 191-200 might also trigger membrane translocation. Plx contains both N-linked (NXS/T) and O-linked (SG) oligosaccharide attachment sites. One of the three potential O-linked sites, residue #586, is flanked by the consensus glycosaminoglycan-linkage sequence for the Xyloside substitution of serine residues in proteoglycans. The Xylosyltransferase recognition sequence is characterized by acidic residues closely followed by Ser-Gly-Xaa-Gly (Zhang, 1996).

With the exception of the above mentioned adhesion motifs and carbohydrate linkage sites, data-base homology comparisons and search programs have failed to place plx in any of the previously described CAM and ECM gene families. However, 20 structurally related proteins of unknown cellular function, expressed in phylogenetically distant organisms, have been identified. The most striking feature shared among these proteins is a similarly positioned novel 74 amino acid domain, which has been named PTM after the first three proteins discovered to contain it: the Drosophila Plx, the human oncoprotein TRE17 (oncoTRE17, Nakamura, 1988), and a human Myeloid cell line expressed protein. Subsequent searches have identified other PTM family members in Arabidopsis thaliana [3 partial cDNA expressed sequence tags (EST)]; an EST from the shoots of oryza sativa (rice); 3 genomic ORFs in Saccharomyces cerevisae; 2 genomic ORFs and 2 ESTs in Caenorhabditis elegans; and in humans, an additional 7 ESTs. Optimal alignment of 10 representative PTMs demonstrates a 50% or greater identity in 83% of the domains' residues. Furthermore, 35% of the positions have 90% or greater identity or similarity. Secondary structure analysis of the PTMs predict a domain with a globular/folded tertiary conformation, comprised of multiple short alpha helixes and beta sheets interrupted by beta turns or structure breaking residues (Zhang, 1996).

One of the conserved blocks of homology within the domain, the pentapeptide Gly-Tyr-Cys-Gln-Gly (GYCQG, positions 51-55), is found in 16 out of the 20 identified PTM domains, and all 20 contain the invariant tripeptide GYC. This identical pentapeptide has been reported in the extracellular domain of the alpha integrin subunit of the human fibronectin receptor (GYCQG, residues 161-165; Argraves, 1987). Related peptides are also found in the alpha integrin subunit extracellular domains of the human vitronectin receptor (GFCQG, residues 152-156; Suzuki, 1987) and the Drosophila PS2 (Inflated, see Myospheroid) antigen (GSCQA, residues 196-200; Bogaert, 1987). Comparisons among PTM proteins whose complete ORFs have been determined (Plx, 732aa; the human oncTRE17, 786aa, and the human myeloid cell line protein, 842aa) reveal that their PTM domains occupy nearly identical positions relative to their predicted amino-termini, starting at residues 141, 138 and 139 for Plx, oncTRE17 and the myeloid protein, respectively. Hydropathicity plot alignments show that Plx and oncTRE17 both have potential membrane spanning hydrophobic regions residing at identical positions relative to their amino-termini. Again, similar to Plx, both the predicted oncTRE17 and myeloid proteins lack an amino-terminus transmembrane signal peptide sequence. All three have multiple proteoglycan binding motifs, glycosylation sites, potential disulfide linkage sites; additionally, the myeloid protein contains the a2b1 integrin binding site Asp-Gly-Glu-Ala (Zhang, 1996 and references).

The striking sequence and predicted structural similarities between PTM proteins suggests that they are functionally related. In man, different PTM encoding transcripts have been found in placenta, infant brain, adult retina, adult heart and in cell lines derived from immature myeloid and T-lymphoblastic leukemia cells. oncTRE17 transcripts, initially isolated from Ewing's sarcoma cells, have now been detected in a wide variety of human cancer cells. Although the cellular distribution of the oncTRE17 protein is currently unknown, its structural homology to Plx suggests that its transforming capacity may be linked to a cell surface signal transduction role. The expression of PTM proteins in organisms as taxonomically dissimilar as plants and man, and in so many different tissues undergoing development is further support for the suggestion that these proteins carry out adhesive roles in all multicellular organisms (Zhang, 1996 and references).

EVOLUTIONARY HOMOLOGS

A common Greenlandic TBC1D4 variant confers muscle insulin resistance and type 2 diabetes

The Greenlandic population, a small and historically isolated founder population comprising about 57,000 inhabitants, has experienced a dramatic increase in type 2 diabetes (T2D) prevalence during the past 25 years. Motivated by this, association mapping of T2D-related quantitative traits was performed in up to 2,575 Greenlandic individuals without known diabetes. Using array-based genotyping and exome sequencing, a nonsense p.Arg684Ter variant (in which arginine is replaced by a termination codon) in the gene TBC1D4 (Drosophila homolog Pollux) with an allele frequency of 17%. Homozygous carriers of this variant have markedly higher concentrations of plasma glucose and serum insulin 2 hours after an oral glucose load compared with individuals with other genotypes (both non-carriers and heterozygous carriers). Furthermore, homozygous carriers have marginally lower concentrations of fasting plasma glucose and fasting serum insulin, and their T2D risk is markedly increased. Heterozygous carriers have a moderately higher plasma glucose concentration 2 hours after an oral glucose load than non-carriers. Analyses of skeletal muscle biopsies showed lower messenger RNA and protein levels of the long isoform of TBC1D4, and lower muscle protein levels of the glucose transporter GLUT4, with increasing number of p.Arg684Ter alleles. These findings are concomitant with a severely decreased insulin-stimulated glucose uptake in muscle, leading to postprandial hyperglycaemia, impaired glucose tolerance and T2D. The observed effect sizes are several times larger than any previous findings in large-scale genome-wide association studies of these traits and constitute further proof of the value of conducting genetic association studies outside the traditional setting of large homogeneous populations (Treebak, 2014).

Identification of novel, evolutionarily conserved Cdc42p-interacting proteins and of redundant pathways linking Cdc24p and Cdc42p to actin polarization in yeast

In the yeast Saccharomyces cerevisiae, Cdc24p functions at least in part as a guanine-nucleotide-exchange factor for the Rho-family GTPase Cdc42p. A genetic screen designed to identify possible additional targets of Cdc24p instead identified two previously known genes, MSB1 and CLA4, and one novel gene, designated MSB3, all of which appear to function in the Cdc24p-Cdc42p pathway. Nonetheless, genetic evidence suggests that Cdc24p may have a function that is distinct from its Cdc42p guanine-nucleotide-exchange factor activity; in particular, overexpression of CDC42 in combination with MSB1 or a truncated CLA4 in cells depleted for Cdc24p allows polarization of the actin cytoskeleton and polarized cell growth, but not successful cell proliferation. MSB3 has a close homologue (designated MSB4) and two more distant homologs (MDR1 and YPL249C) in S. cerevisiae and also has homologs in Schizosaccharomyces pombe, Drosophila (pollux), and humans (the oncogene tre17). Deletion of either MSB3 or MSB4 alone does not produce any obvious phenotype, and the msb3 msb4 double mutant is viable. However, the double mutant grows slowly and has a partial disorganization of the actin cytoskeleton, but not of the septins, in a fraction of cells that are larger and rounder than normal. Like Cdc42p, both Msb3p and Msb4p localize to the presumptive bud site, the bud tip, and the mother-bud neck, and this localization is Cdc42p dependent. Taken together, the data suggest that Msb3p and Msb4p may function redundantly downstream of Cdc42p, specifically in a pathway leading to actin organization. From previous work, the BNI1, GIC1, and GIC2 gene products also appear to be involved in linking Cdc42p to the actin cytoskeleton. Synthetic lethality and multicopy suppression analyses among these genes, MSB, and MSB4, suggest that the linkage is accomplished by two parallel pathways, one involving Msb3p, Msb4p, and Bni1p, and the other involving Gic1p and Gic2p. The former pathway appears to be more important in diploids and at low temperatures, whereas the latter pathway appears to be more important in haploids and at high temperatures (Bi, 2000).

Molecular cloning of a cDNA with a novel domain present in the tre-2 oncogene and the yeast cell cycle regulators BUB2 and cdc16

A protein related to Plx is TBC1 (Richardson, 1995), a mouse protein which had homology to the majority of Plx (Xu, 1998). In an effort to identify genes that are differentially regulated during mast cell development, subtracted cDNA prepared from wild-type murine P815 mastocytoma cells and a P815 subline that exhibits properties of mast cell differentiation was used to screen mast cell cDNA libraries. Several known mast cell-specific cDNAs were isolated including mast cell carboxypeptidase A (MC-CPA), murine mast cell protease-5 (MMCP-5), and gp49. A novel cDNA, designated Tbc1, was identified that shows differential expression in the two mast cell lines. The amino acid sequence predicted from the cDNA contains a 200 amino acid domain that is homologous to regions in the tre-2 oncogene and yeast regulators of mitosis BUB2 and cdc16. The N-terminal region contains a number of cysteine and histidine residues, potentially encoding a zinc finger domain. Tbc1 is a nuclear protein and is expressed in highest levels in hematopoietic cells, testis and kidney. Within these tissues, expression of Tbc1 is cell- and stage-specific. Based on sequence similarity, pattern of expression and subcellular localization, Tbc1 may play a role in the cell cycle and differentiation of various tissues (Richardson, 1995).

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pollux: Regulation | Developmental Biology | Effects of Mutation | References

date revised: 5 April 2021

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