Persistence of duplicated PAC1 receptors in the teleost, Sparus auratus.

BACKGROUND: : Duplicated genes are common in vertebrate genomes. Their persistence is assumed to be either a consequence of gain of novel function (neofunctionalisation) or partitioning of the function of the ancestral molecule (sub-functionalisation). Surprisingly few studies have evaluated the extent of such modifications despite the numerous duplicated receptor and ligand genes identified in vertebrate genomes to date. In order to study the importance of function in the maintenance of duplicated genes, sea bream (Sparus auratus) PAC1 receptors, sequence homologues of the mammalian receptor specific for PACAP (Pituitary Adenylate Cyclase-Activating Polypeptide), were studied. These receptors belong to family 2 GPCRs and most of their members are duplicated in teleosts although the reason why both persist in the genome is unknown. RESULTS: : Duplicate sea bream PACAP receptor genes (sbPAC1A and sbPAC1B), members of family 2 GPCRs, were isolated and share 77% amino acid sequence identity. RT-PCR with specific primers for each gene revealed that they have a differential tissue distribution which overlaps with the distribution of the single mammalian receptor. Furthermore, in common with mammals, the teleost genes undergo alternative splicing and a PAC1Ahop1 isoform has been characterised. Duplicated orthologous receptors have also been identified in other teleost genomes and their distribution profile suggests that function may be species specific. Functional analysis of the paralogue sbPAC1s in Cos7 cells revealed that they are strongly stimulated in the presence of mammalian PACAP27 and PACAP38 and far less with VIP (Vasoactive Intestinal Peptide). The sbPAC1 receptors are equally stimulated (LOGEC50 values for maximal cAMP production) in the presence of PACAP27 (-8.74 +/- 0.29 M and -9.15 +/- 0.21 M, respectively for sbPAC1A and sbPAC1B, P > 0.05) and PACAP38 (-8.54 +/- 0.18 M and -8.92 +/- 0.24 M, respectively for sbPAC1A and sbPAC1B, P > 0.05). Human VIP was found to stimulate sbPAC1A (-7.23 +/- 0.20 M) more strongly than sbPAC1B (-6.57 +/- 0.14 M, P < 0.05) and human secretin (SCT), which has not so far been identified in fish genomes, caused negligible stimulation of both receptors. CONCLUSION: : The existence of functionally divergent duplicate sbPAC1 receptors is in line with previously proposed theories about the origin and maintenance of duplicated genes. Sea bream PAC1 duplicate receptors resemble the typical mammalian PAC1, and PACAP peptides were found to be more effective than VIP in stimulating cAMP production, although sbPAC1A was more responsive for VIP than sbPAC1B. These results together with the highly divergent pattern of tissue distribution suggest that a process involving neofunctionalisation occurred after receptor duplication within the fish lineage and probably accounts for their persistence in the genome. The characterisation of further duplicated receptors and their ligands should provide insights into the evolution and function of novel protein-protein interactions associated with the vertebrate radiation.

The novel inhibitory receptor G6B is expressed on the surface of platelets and attenuates platelet function in vitro.

The G6B cell-surface receptor, which contains a single Ig-like domain, has been shown to bind to SHP-1 and SHP-2 after phosphorylation of 2 immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic tail, classifying this protein as a new member of the family of inhibitory receptors. In this study, we demonstrate by real-time polymerase chain reaction (PCR) and Western-blot analysis that G6B is expressed on platelets. Cross-linking of G6B with polyclonal antisera has a significant inhibitory effect on platelet aggregation and activation by agonists such as ADP and collagen-related peptide (CRP). This inhibitory function of G6B appears to operate in a calcium-independent manner. Our results suggest that G6B represents a novel inhibitory receptor found on the surface of platelets and that it could be an antithrombotic drug target.

The cell surface receptor G6b, a member of the immunoglobulin superfamily, binds heparin.

The G6b gene, located in the human Major Histocompatibility Complex, encodes a receptor of the immunoglobulin (Ig) superfamily. In this study, we show using a variety of techniques that the extracellular domain of the G6b protein, containing a single Ig-like domain, binds to heparin with high affinity. In an ELISA assay, this binding was displaceable with soluble heparin with an IC50 value of approximately 0.5 microg/ml. Other sulfated glycans showed weaker or no competition. The observed interaction between G6b and heparin is strongly salt dependent suggesting a mainly electrostatic interaction. Heparin might modulate the interaction of G6b with its as yet unidentified protein ligand.

Adaptor signalling proteins Grb2 and Grb7 are recruited by human G6f, a novel member of the immunoglobulin superfamily encoded in the MHC.

The human G6f protein, which is encoded by a gene in the MHC, is a putative cell-surface receptor belonging to the immunoglobulin superfamily. The intracellular tail of G6f is 40 amino acids in length and contains one tyrosine residue (Y281), which is phosphorylated after treatment of cells with pervanadate. This tyrosine residue is found in a consensus-binding motif (YXN) for the Src homology 2 domains of Grb2 and Grb7 (where Grb stands for growth-factor-receptor-bound protein). Glutathione S-transferase pull-down assays showed that the interaction of G6f with both Grb2 and Grb7 is mediated through the Src homology 2 domains of these two proteins and is dependent on the phosphorylation of G6f. Immunoprecipitation experiments showed the interaction of full-length phosphorylated G6f with both full-length Grb2 and Grb7. Antibody cross-linking of G6f expressed in K562 cells resulted in a transient phosphorylation of p42/44 MAP kinase (also known as extracellular-signal-regulated protein kinase-1/2; MAP stands for mitogen-activated protein) which could be prevented by MAP kinase kinase (MEK) inhibitors. These results suggest a coupling of G6f with downstream signal transduction pathways involving Grb2 and Grb7, including the Ras-MAP kinase pathway.