Identification of novel splice variants of Adhesion G protein-coupled receptors.

Alternative splicing is an important mechanism to generate proteome diversity in higher eukaryotic organisms. We searched for splice variants of the human Adhesion family of G protein-coupled receptors (GPCRs) using mRNA sequences and expressed sequence tags. The results presented here describe 53 human splice variants among the 33 Adhesion GPCRs. Many of these variants appear to be coding for "functional" proteins (29) while the others are seemingly "non-functional" (24). Novel functional splice variants were found for: CD97, CELR3, EMR2, EMR3, GPR56, GPR110, GPR112-GPR114, GPR116, GPR123-GPR126, GPR133, HE6, and LEC1-LEC3. Splice variants for GPR116, GPR125, GPR126, and HE6 were found conserved in other species. Several of the functional splice variants lack one or more of the functional domains that are found in the N-termini of these receptors. These functional domains are likely to affect ligand binding or interaction with other proteins and these novel splice variants may have important roles for the specificity of interactions between these receptors and extracellular molecules. Another type of splice variants found here lacks a GPCR proteolytic site (GPS). The GPS domain has been shown to be essential for the proteolytic cleavage of the receptors N-termini and for cellular surface expression. We suggest that these alternative splice variants may be crucial for the function of the receptors while the seemingly non-functional splice variants may be a part of a regulative mechanism.

Comprehensive repertoire and phylogenetic analysis of the G protein-coupled receptors in human and mouse.

Understanding differences in the repertoire of orthologous gene pairs is vital for interpretation of pharmacological and physiological experiments if conclusions are conveyed between species. Here we present a comprehensive dataset for G protein-coupled receptors (GPCRs) in both human and mouse with a phylogenetic road map. We performed systematic searches applying several search tools such as BLAST, BLAT, and Hidden Markov models and searches in literature data. We aimed to gather a full-length version of each human or mouse GPCR in only one copy referring to a single chromosomal position. Moreover, we performed detailed phylogenetic analysis of the transmembrane regions of the receptors to establish accurate orthologous pairs. The results show the identity of 495 mouse and 400 human functional nonolfactory GPCRs. Overall, 329 of the receptors are found in one-to-one orthologous pairs, while 119 mouse and 31 human receptors originate from species-specific expansions or deletions. The average percentage similarity of the orthologue pairs is 85%, while it varies between the main GRAFS families from an average of 59 to 94%. The orthologous pairs for the lipid-binding GPCRs had the lowest levels of conservation, while the biogenic amines had highest levels of conservation. Moreover, we searched for expressed sequence tags (ESTs) and identified more than 17,000 ESTs matching GPCRs in mouse and human, providing information about their expression patterns. On the whole, this is the most comprehensive study of the gene repertoire that codes for human and mouse GPCRs. The datasets are available for downloading.

The gene repertoire and the common evolutionary history of glutamate, pheromone (V2R), taste(1) and other related G protein-coupled receptors.

Glutamate receptors (also known as clan C) are one of the main groups of GPCRs with many subgroup linked through complex evolutionary relationships. We performed thorough searches for genes coding for proteins belonging to this family in the human, mouse, Fugu, and zebrafish genomes, as well as in four invertebrate species. We assembled over 70 new full-length sequences from protein predictions excluding pseudogenes. This resulted in a total of 22 full-length sequences from the human genome, 79 from the mouse genome, 30 from the Fugu genome, and 32 from the zebrafish genome (pseudogenes are not included in these numbers). We show that the vertebrate Glutamate GPCRs form four main phylogenetic groups with a total of eight subgroups (Group I: V2R, TAS1R, GPRC6A, and CASR, Group II: GRM, Group III: GABA together with previously unpublished GPR158 and GPR158L and Group IV: GPRC5). All eight receptor subgroups are present both in mammals and fish, except for GPRC5 and GPR158. The pheromone (V2R), GPRC6, and sweet taste (TAS1) receptors were not found in invertebrates while GRM, GABA, and CASR were found in both C. elegans and C. intestinalis. The pheromone receptors are found in high numbers in mouse, zebrafish and Fugu but are only found as pseudogenes in the human genome. This report provides a comprehensive overview of the expansion/deletions of the groups within the Glutamate receptor family.

Origin of the prolactin-releasing hormone (PRLH) receptors: evidence of coevolution between PRLH and a redundant neuropeptide Y receptor during vertebrate evolution.

We present seven new vertebrate homologs of the prolactin-releasing hormone receptor (PRLHR) and show that these are found as two separate subtypes, PRLHR1 and PRLHR2. Analysis of a number of vertebrate sequences using phylogeny, pharmacology, and paralogon analysis indicates that the PRLHRs are likely to share a common ancestry with the neuropeptide Y (NPY) receptors. Moreover, a micromolar level of NPY was able to bind and inhibit completely the PRLH-evoked response in PRLHR1-expressing cells. We suggest that an ancestral PRLH peptide started coevolving with a redundant NPY binding receptor, which then became PRLHR, approximately 500 million years ago. The PRLHR1 subtype was shown to have a relatively high evolutionary rate compared to receptors with fixed peptide preference, which could indicate a drastic change in binding preference, thus supporting this hypothesis. This report suggests how gene duplication events can lead to novel peptide ligand/receptor interactions and hence spur the evolution of new physiological functions.

The phylogenetic relationship of the glutamate and pheromone G-protein-coupled receptors in different vertebrate species.

Searches in genomic databases for human, mouse, zebrafish, and pufferfish genes resulted in the identification of more than 180 protein predictions belonging to the glutamate family of G-protein-coupled receptors (GPCRs). Comparison of data sets from the different species showed that most of the receptor subgroups that form the glutamate family are present in both mammalian and bony fish lineage. This finding indicates that these groups share a phylogenetically ancient origin. The present study also shows that the pheromone-receptor subgroup has undergone independent expansions in three of the four species, leaving the human genome completely deprived of all pheromone receptors.

The repertoire of trace amine G-protein-coupled receptors: large expansion in zebrafish.

Trace amines, such as tyramine, beta-phenylethylamine, tryptamine, and octopamine, are present in trace levels in nervous systems and bind a specific family of G-protein-coupled receptors (GPCR), but the function or origin of this system is not well understood. We searched the genomes of several eukaryotic species for receptors similar to the mammalian trace amine (TA) receptor subfamily. We identified 18 new receptors in rodents that are orthologous to the previously known TA-receptors. Remarkably, we found 57 receptors (and 40 pseudogenes) of this type in the zebrafish (Danio rerio), while fugu (Takifugu rubripes) had only eight receptors (and seven pseudogenes). We mapped 47 of the zebrafish TA-receptors on chromosomes using radiation hybrid panels and meiotic mapping. The results, together with the degree of conservation and phylogenetic relationships displayed among the zebrafish receptors suggest that the family arose through several different mechanisms involving tetraploidization, block duplications, and local duplication events. Interestingly, these vertebrate TA-receptors do not show a close evolutionary relationship to the invertebrate TA-binding receptors in fruitfly (Drosophila melanogaster), indicating that the ability to bind TA have evolved at least twice in animal evolution. We collected in total over 100 vertebrate TA-receptor sequences, and our phylogenetic analysis shows that several TA-receptors have evolved rapidly with remarkable species variation and that the common ancestor of vertebrate TA-receptors arose before the split of the ray-finned and lobe-finned fishes. The evolutionary history of the TA-receptors is more complex than for most other GPCR families and here we suggest a mechanism by which they may have arisen.

The human and mouse repertoire of the adhesion family of G-protein-coupled receptors.

The adhesion G-protein-coupled receptors (GPCRs) (also termed LN-7TM or EGF-7TM receptors) are membrane-bound proteins with long N-termini containing multiple domains. Here, 2 new human adhesion-GPCRs, termed GPR133 and GPR144, have been found by searches done in the human genome databases. Both GPR133 and GPR144 have a GPS domain in their N-termini, while GPR144 also has a pentraxin domain. The phylogenetic analyses of the 2 new human receptors show that they group together without close relationship to the other adhesion-GPCRs. In addition to the human genes, mouse orthologues to those 2 and 15 other mouse orthologues to human were identified (GPR110, GPR111, GPR112, GPR113, GPR114, GPR115, GPR116, GPR123, GPR124, GPR125, GPR126, GPR128, LEC1, LEC2, and LEC3). Currently the total number of human adhesion-GPCRs is 33. The mouse and human sequences show a clear one-to-one relationship, with the exception of EMR2 and EMR3, which do not seem to have orthologues in mouse. EST expression charts for the entire repertoire of adhesion-GPCRs in human and mouse were established. Over 1600 ESTs were found for these receptors, showing widespread distribution in both central and peripheral tissues. The expression patterns are highly variable between different receptors, indicating that they participate in a number of physiological processes.