InteractiveFly: GeneBrief

CUB and LDLa domain: Biological Overview | References

Endocytosis of G-protein-coupled receptors (GPCRs) and associated channels contributes to desensitization and adaptation of a variety of signaling cascades. In Drosophila melanogaster, the main light-sensing rhodopsin (Rh1; encoded by ninaE) and the downstream ion channel, transient receptor potential like (TRPL), are endocytosed in response to light, but the mechanism is unclear. By using an RNA-Sequencing (RNA-Seq) approach, a protein was discovered that was named CULD, a photoreceptor-cell enriched CUB- and LDLa-domain transmembrane protein, that is required for endocytic trafficking of Rh1 and TRPL. CULD localized to endocytic Rh1-positive or TRPL-positive vesicles. Mutations in culd resulted in the accumulation of Rh1 and TRPL within endocytic vesicles, and disrupted the regular turnover of endocytic Rh1 and TRPL. In addition, loss of CULD induced light- and age-dependent retinal degeneration, and reduced levels of Rh1, but not of TRPL, suppressed retinal degeneration in culd-null mutant flies. These data demonstrate that CULD plays an important role in the endocytic turnover of Rh1 and TRPL, and suggest that CULD-dependent rhodopsin endocytic trafficking is required for maintaining photoreceptor integrity (Xu, 2016).

G-protein-coupled receptors (GPCRs) are the largest family of membrane receptors and, therefore, transduce signals from a wide variety of hormones, cytokines, neurotransmitters, as well as sensory stimuli. Each of these interactions triggers distinct intracellular responses through heterotrimeric G proteins. Upon continuous stimulation, GPCRs are deactivated by arrestins, and internalized through dynamin-dependent endocytosis. Many internalized GPCRs undergo lysosomal degradation and/or recycling, leading to downregulation of receptor levels, which is important for reducing the strength and duration of cellular responsiveness following various stimuli (Xu, 2016).

The Drosophila phototransduction cascade is a model pathway for the dissection of GPCR signaling and associated regulatory processes. Proteins of the visual signal transduction cascade are found within rhabdomeres, which are specialized compartments within photoreceptor cells that contain tightly packed microvilli. Light-induced activation of rhodopsin triggers the phototransduction cascade by stimulating the vision protein phospholipase C, which is encoded by the no receptor potential A (norpA) gene, through the α subunit of the heterotrimeric G protein DGq. This opens the transient receptor potential (TRP) channel and the TRP-like (TRPL) Ca²⁺/cation channel, and depolarizes the photoreceptor neurons. Meanwhile, activated rhodopsin, which is referred to as metarhodopsin, is immediately bound by arrestin and deactivated. After inactivation, metarhodopsin is either photoconverted back into rhodopsin or internalized for degradation. Although the majority of internalized metarhodopsin is degraded, with newly synthesized rhodopsin replenishing the pool, it has recently been reported that internalized rhodopsin (Rh1; encoded by ninaE in Drosophila melanogaster) can be recycled upon stimulation with light. The principle arrestin, Arr2, plays a pivotal role in deactivating rhodopsin, whereas Arr1 binds and internalizes rhodopsin (Xu, 2016).

Long-term adaptation to light stimuli also involves the dynamic activity-dependent translocation of signaling proteins that are not GPCRs. As seen with mammalian Rod photoreceptors, light induces the movement of Arr2 and Arr1 into the rhabdomeres. In Drosophila, TRP and TRPL function as the primary light-activated channels. TRP stably localizes to the rhabdomeres by forming a multiprotein signaling complex, the signalplex with inactivation-no-after-potential D protein (INAD), a protein that contains five PDZ domains. In contrast, illumination results in TRPL translocating from the rhabdomeres to an intracellular storage compartment within the cell body. However, the mechanisms that underlie light-induced translocation and trafficking of rhodopsin and TRPL are not yet fully understood. Furthermore, it is unclear whether this endocytic trafficking of TRPL plays a physiological role in maintaining the integrity of photoreceptor cells (Xu, 2016).

By using an RNA-Sequencing (RNA-Seq) approach, this study identified a so-far-unknown gene that is enriched in photoreceptors, and encodes a transmembrane protein with both a CUB and an LDLa domain. This protein was named CULD (CUB- and LDLa-domain protein). CULD mainly localized to the endocytic TRPL- or Rh1-positive vesicles. Mutations in culd led to endosomal accumulation of Rh1 and TRPL, which disrupted the light sensitivity of photoreceptors; blocking of Arr1-mediated endocytosis eliminated the intracellular accumulation of Rh1. Moreover, culd mutants underwent light-dependent retinal degeneration, and resulted in a phenotype that could be rescued by reducing the levels of Rh1. These data indicate that CULD is essential for the function and survival of photoreceptor cells by promoting the endocytic turnover of Rh1 and TRPL (Xu, 2016).

A microarray analysis has previously been used to compare the genes expressed in wild-type heads with heads from a mutant fly that lacked eyes in order to identify eye-enriched genes, which led to the further identification of some genes functioning in phototransduction. However, owing to multiple cell types in the compound eye, many genes identified in this analysis might not function in photoreceptor cells. This study describes an RNA-Seq screen to identify genes expressed predominantly in photoreceptors. Among the 58 genes identified, 36 genes were known to function in photoreceptor cells, representing most of the genes that play major roles in phototransduction or retinal degeneration. However, 22 genes had not been described as being enriched in photoreceptor cells previously. Among them, cg9935 (Eye-enriched kainate receptor: Ekar) has been recently reported to regulate the retrograde glutamate signal in photoreceptor cells and contribute to light-evoked depolarization during phototransduction (Hu, 2015). This study further characterized the new photoreceptor cell-enriched gene culd as being required for turnover of Rh1 and TRPL. Although culd had also been identified as an eye-enriched gene in the earlier microarray analysis that compared RNA expression in wild-type and eyeless heads, 93 other eye-enriched candidates prevented focusing on CULD (Xu, 2004). In this RNA-Seq screen, only photoreceptor-cell-enriched genes can be identified, and a reasonable number of candidates might represent new factors functioning in phototransduction. However, some eye-enriched genes important for phototransduction might be missed in this screen. For example, recently identified polyglutamine-binding protein 1 (PQBP1) was not found as a photoreceptor-cell-specific gene in the RNA-Seq screen. This might be because PQBP1 is also expressed in other non-photoreceptor retinal cells. Overall, this screen for photoreceptor-enriched genes sheds a light on further understanding of phototransduction and mechanisms of retinal degeneration (Xu, 2016).

Appropriate signals cause arrestins to translocate to the plasma membrane where they bind to activated GPCRs, thereby inhibiting G-protein-dependent signaling and regulating GPCR endocytosis and trafficking. In Drosophila there are two arrestins within photoreceptors, Arr1 and Arr2. Although Arr2 binds to Rh1, it is Arr1 that primarily colocalizes with Rh1 in internalized vesicles. Therefore, Arr1 might mediate light-dependent endocytosis of Rh1, whereas Arr2 functions to quench activated Rh1. In culd mutant flies, Rh1 was immobilized within endocytic vesicles and Arr1 colocalized with the endocytic Rh1; blocking the Arr1-medicated endocytosis in culd mutant cells eliminated the abnormal intracellular accumulation of Rh1. These data strongly suggest that CULD functions downstream of Arr1-mediated endocytosis of Rh1 (Xu, 2016).

Early endosomes containing Rab5 serve as a focal point of the endocytic pathway. Sorting events initiated in early endosomes determine the subsequent fate of internalized proteins, that is, whether they will be recycled to the plasma membrane or degraded within lysosomes. Rh1 and TRPL share the same internalization pathway, and during light stimulation Rab5 initially mediates this vesicular transport pathway. In wild-type photoreceptors, however, Rh1 and TRPL have different fates from common Rab5-positive early endosomes. The majority of Rh1 is eventually delivered to lysosomes for degradation, whereas most internalized TRPL tends to be stored. In wild-type cells, the photoreceptor-enriched protein CULD colocalized with the endocytic TRPL or Rh1 vesicles, and the majority of CULD-positive vesicles were also Rab5-positive. This spatial pattern indicates that CULD is required for the endocytic trafficking of TRPL and Rh1 after they are internalized (Xu, 2016).

CULD functions during the early steps of endocytosis that immediately follow internalization, which is a pathway involved in rhodopsin and TRPL endocytic turnover. Eliminating CULD had profound effects on the photoreceptor physiology. In both vertebrates and invertebrates, the light sensitivity of photoreceptor cells is primarily determined by functional rhodopsin. The culd mutant flies exhibited a gradual reduction in light sensitivity, which suggests that the amount of functional rhodopsin is reduced in culd mutant flies. As the amount of the monomer form of Rh1 was not affected and a large fraction of Rh1 accumulated within intracellular vesicles in culd mutant photoreceptor cells, the rhabdomeral Rh1 levels might be reduced. It is also likely that the endocytic degradation of Rh1 scavenges damaged Rh1 molecules, and blocking this process might lead to the accumulation of dysfunctional Rh1 in rhabdomeres (Xu, 2016).

TRPL has been reported to translocate from rhabdomeres to intracellular compartments for storage during prolonged light stimulation. However, a recent study suggests that some endocytic TRPL proteins are also delivered to lysosomes for degradation (Cerny, 2013). Mutations in culd impaired TRPL endocytic trafficking upon light stimulation, leading to the retention of TRPL in Rab7-positive vesicles. TRPL protein levels were increased in culd mutants and this is probably due to decreased TRPL degradation (Xu, 2016).

As a major light sensor within photoreceptor cells, a small amount of activated Rh1 is internalized and degraded upon light stimulation. This is followed by replenishment of the rhabdomeric Rh1 pool. Therefore, a balance between Rh1 endocytosis and replenishment is required for Rh1 homeostasis under light conditions. Prolonged exposure to blue light triggers massive endocytosis of Rh1 and leads to a gradual loss of Rh1. Mutations in culd blocked Rh1 degradation during prolonged light treatment, indicating that the loss of CULD inhibited the Rh1-degradation pathway. Unlike TRPL, Rh1 levels were not increased, which suggests that Rh1 replenishment is strictly controlled. Given that, in Drosophila, rhodopsin levels are regulated by both the synthesis of the opsin and the chromophore subunits, it might be reasonable that in culd mutant cells, the chromophore is not released from the accumulated rhodopsin, and the reduction of free retinal pool might limit the synthesis of new rhodopsin (Xu, 2016).

Both vertebrates and invertebrates have a family of transmembrane proteins that contain both CUB- and LDLa- domains. However, only a few CUB/LDLa proteins have been functionally characterized. Among these proteins, NETO1 and NETO2 (see Drosophila Neto) have been intensively studied. NETO1 functions as an auxiliary subunit of ionotropic glutamate receptors, N-methyl-D-aspartate receptors and kainate receptors, modulating the channel properties of these glutamate receptors (Ng, 2009; Straub, 2011; Tang, 2011; Zhang, 2009). NETO2 maintains normal levels of the neuron-specific K+-Cl- co-transporter KCC2 (also known as SLC12A5), and loss of NETO2-KCC2 interactions reduces KCC2-mediated Cl- extrusion, and decreases synaptic inhibition in hippocampal neurons (Ivakine, 2013). Moreover, in both Drosophila and C. elegans, the CUB/LDLa proteins NETO and SOL-2 are required for the clustering and functioning of glutamine receptors, thereby contributing to neuronal signaling pathways (Kim, 2012; Wang, 2012). This study cloned a new gene culd, which encodes a member of the CUB/LDLa family proteins specifically expressed in the Drosophila photoreceptor cell; this protein containing a CUB domain, an LDLa domain and one predicted transmembrane motif. CULD was not directly required for the activity of receptors or channels, but instead mediated the endocytic trafficking of Rh1 and TRPL. Loss of CULD led to the accumulation of Rh1 and TRPL in endocytic vesicles, and subsequent retinal degeneration. Therefore, this study revealed a new function of the CUB/LDLa family proteins, namely the endocytic turnover of receptors and channels (Xu, 2016).

It has been proposed that the accumulation of Rh1-Arr2 complexes in late endosomes triggers cell death of photoreceptor cells. Internalized Rh1-Arr2 complexes are not degraded but instead accumulate in late endosomes of norpA mutant photoreceptor cells. Similar rhodopsin accumulations are seen in mutations that affect the trafficking of late endosomes to lysosomes, which causes light-dependent retinal degeneration. Toxic Rh1-Arr2 complexes also induce retinal degeneration in rdgC, rdgB and fatp mutant flies. The culd mutations caused the accumulation of Rh1-Arr1 complexes and TRPL in endocytic vesicles and light-dependent retinal degeneration, suggesting that endosomal accumulation of either channels or receptors induced cell death. In addition, the evidence that ninaE^P332 but not trpl³⁰² rescued photoreceptor degeneration of the culd¹ mutants suggests that abnormal Rh1-Arr1 accumulation induces cell degeneration, whereas intracellular accumulation of TRPL does not contribute to the neuronal degeneration (Xu, 2016).

Search PubMed for articles about Drosophila Culd

Cerny, A. C., Oberacker, T., Pfannstiel, J., Weigold, S., Will, C. and Huber, A. (2013). Mutation of light-dependent phosphorylation sites of the Drosophila transient receptor potential-like (TRPL) ion channel affects its subcellular localization and stability. J Biol Chem 288(22): 15600-15613. PubMed ID: 23592784

Hu, W., Wang, T., Wang, X. and Han, J. (2015). Ih channels control feedback regulation from amacrine cells to photoreceptors. PLoS Biol 13(4): e1002115. PubMed ID: 25831426

Ivakine, E. A., Acton, B. A., Mahadevan, V., Ormond, J., Tang, M., Pressey, J. C., Huang, M. Y., Ng, D., Delpire, E., Salter, M. W., Woodin, M. A. and McInnes, R. R. (2013). Neto2 is a KCC2 interacting protein required for neuronal Cl- regulation in hippocampal neurons. Proc Natl Acad Sci U S A 110(9): 3561-3566. PubMed ID: 23401525

Kim, Y. J., Bao, H., Bonanno, L., Zhang, B. and Serpe, M. (2012). Drosophila Neto is essential for clustering glutamate receptors at the neuromuscular junction. Genes Dev 26(9): 974-987. PubMed ID: 22499592

Ng, D., Pitcher, G. M., Szilard, R. K., Sertie, A., Kanisek, M., Clapcote, S. J., Lipina, T., Kalia, L. V., Joo, D., McKerlie, C., Cortez, M., Roder, J. C., Salter, M. W. and McInnes, R. R. (2009). Neto1 is a novel CUB-domain NMDA receptor-interacting protein required for synaptic plasticity and learning. PLoS Biol 7(2): e41. PubMed ID: 19243221

Straub, C., Hunt, D. L., Yamasaki, M., Kim, K. S., Watanabe, M., Castillo, P. E. and Tomita, S. (2011). Distinct functions of kainate receptors in the brain are determined by the auxiliary subunit Neto1. Nat Neurosci 14(7): 866-873. PubMed ID: 21623363

Tang, M., Pelkey, K. A., Ng, D., Ivakine, E., McBain, C. J., Salter, M. W. and McInnes, R. R. (2011). Neto1 is an auxiliary subunit of native synaptic kainate receptors. J Neurosci 31(27): 10009-10018. PubMed ID: 21734292

Wang, R., Mellem, J. E., Jensen, M., Brockie, P. J., Walker, C. S., Hoerndli, F. J., Hauth, L., Madsen, D. M. and Maricq, A. V. (2012). The SOL-2/Neto auxiliary protein modulates the function of AMPA-subtype ionotropic glutamate receptors. Neuron 75(5): 838-850. PubMed ID: 22958824

Xu, H., Lee, S. J., Suzuki, E., Dugan, K. D., Stoddard, A., Li, H. S., Chodosh, L. A. and Montell, C. (2004). A lysosomal tetraspanin associated with retinal degeneration identified via a genome-wide screen. EMBO J 23(4): 811-822. PubMed ID: 14963491

Xu, Y. and Wang, T. (2016). CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells. J Cell Sci 129(2): 394-405. PubMed ID: 26598556

Zhang, W., St-Gelais, F., Grabner, C. P., Trinidad, J. C., Sumioka, A., Morimoto-Tomita, M., Kim, K. S., Straub, C., Burlingame, A. L., Howe, J. R. and Tomita, S. (2009). A transmembrane accessory subunit that modulates kainate-type glutamate receptors. Neuron 61(3): 385-396. PubMed ID: 19217376

date revised: 10 February 2017

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