Receptor oligomerization: from early evidence to current understanding in class B GPCRs

No abstract

The P2Y receptor family represents a wide-ranging set of G-protein-coupled receptors that respond to purinergic ligands, and are involved in many physiological processes. From signalling assays in tsA201 cells, the Kennedy lab has previously proposed the formation of a constitutive heterodimer between co-expressed human P2Y1 and P2Y12 receptors; this could represent a _target for the control of pain sensing neurones. Therefore, it was aimed in this project to characterise if the receptors physically interacted using co-immunoprecipitation, and also to investigate the endogenous expression of other potentially interacting hP2Y receptors in the tsA201 cell line. In this study, tsA201 cells were determined to express mRNA for hP2Y1, 2, 4, 6, 12, 14 receptors using reverse transcriptase PCR and confirmatory Sanger sequencing. Furthermore, the cells demonstrated Ca2+ responses to all of the natural hP2Y receptor agonists tested, the order of potency being ADP > ATP > UTP > UDP. When co-expressing hP2Y1 and hP2Y12 receptors oppositely tagged with a HA or fluorescent protein (FP), immunoprecipitating for the HA tag and blotting for the FP showed a visible high molecular weight protein complex of ~130 to >250 KDa for either combination of receptors. This implied that the FP tagged receptor had co-immunoprecipitated with the other HA tagged receptor as this protein was also visible when blotting for the HA tag, signifying the presence of both tagged receptors in the complex. These results were overall suggestive of functional hP2Y1, 2, 12 receptor expression in the tsA201 cell line, and possibly also hP2Y4, 6, 14 receptors. The high molecular weight protein complex detected could represent a constitutive hP2Y1-hP2Y12 receptor heterodimer as proposed. Whilst it was not possible to specifically either confirm or refute the physical formation of the heterodimer with the data acquired, it was potentially supportive, and furthermore suggested other future paths of investigation.

In mammals, secretin is a 27-amino acid peptide that was first studied in 1902 by Bayliss and Starling from the extracts of the jejunal mucosa for its ability to stimulate pancreatic secretion. To date, secretin has only been identified in tetrapods, with the earliest diverged secretin found in frogs. Despite being the first hormone discovered, secretin's evolutionary origin remains enigmatic, it shows moderate sequence identity in nonmammalian tetrapods but is highly conserved in mammals. Current hypotheses suggest that although secretin has already emerged before the divergence of osteichthyans, it was lost in fish and retained only in land vertebrates. Nevertheless, the cognate receptor of secretin has been identified in both actinopterygian fish (zebrafish) and sarcopterygian fish (lungfish). However, the zebrafish secretin receptor was shown to be nonbioactive. Based on the present information that the earliest diverged bioactive secretin receptor was found in lungfish, and its ability to interact with both vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide potently suggested that secretin receptor was descended from a VPAC-like receptor gene before the Actinopterygii-Sarcopterygii split in the vertebrate lineage. Hence, secretin and secretin receptor have gone through independent evolutionary trajectories despite their concurrent emergence post-2R. A functional secretin-secretin receptor axis has probably emerged in the amphibians. Although the pleiotropic actions of secretin are well documented in the literature, only limited information of its physiological functions in nonmammalian tetrapods have been reported. To decipher the structural and functional divergence of secretin and secretin receptor, functional characterization of the ligandreceptor pair in nonmammals would be the next perspective for investigation.