The Interactive Fly
Zygotically transcribed genes

G-protein coupled receptors - a classification based on primary structure and function

A genome wide survey of Drosophila G-protein coupled receptors

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

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

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


Notes:


A genome wide survey of Drosophila 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. 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 and CG13803 responds specifically to the FMRF-amide-related peptide dromyosuppressin. 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 Gi-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).


References

Brody, T. and Cravchik, A. (2000). Drosophila melanogaster G protein-coupled receptors. J. Cell Bio. 150: F83-88. 10908591

Johnson, E. C., et al. (2003). Identification of Drosophila neuropeptide receptors by G protein-coupled receptors-{beta}-Arrestin2 interactions. J. Biol. Chem. 278 (52): 52172-52178. 14555656

Pascall, J. C. and Brown, K. D. (1998). Characterization of a mammalian cDNA encoding a protein with high sequence similarity to the Drosophila regulatory protein Rhomboid. FEBS Lett. 429(3): 337-340. Medline abstract: 9662444

Park, Y., Kim, Y. J. and Adams, M. E. (2002b). Identification of G protein-coupled receptors for Drosophila PRXamide peptides, CCAP, corazonin, and AKH supports a theory of ligand-receptor coevolution. Proc. Natl. Acad. Sci. 99: 11423-11428. 12177421

Rizzo, M. J., Evans, J. P., Burt, M., Saunders, C. J. and Johnson, E. C. (2018). Unexpected role of a conserved domain in the first extracellular loop in G protein-coupled receptor trafficking. Biochem Biophys Res Commun 503(3): 1919-1926. PubMed ID: 30064912

Wasserman, J. D., et al. (2000). A family of rhomboid-like genes: Drosophila rhomboid-1 and roughoid/rhomboid-3 cooperate to activate EGF receptor signaling. Genes Dev. 14: 1651-1663. Medline abstract: 10887159




Zygotically transcribed genes

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