Structure And Functions Of Signal-transducing G-proteins

Deafness &Other Communication Disorders

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Abstract

We extended our work on the human 5-HT receptors to understand the apparent discrepancies among investigations determining G-protein selectivity for GPCR structures in heterologous cell expression models. To examine this we compared the apparent G-protein selectivity for two human 5-HT receptors, 5HT2c and 5HT1A, activating Gq and Gi respectively. Examined by co-expression of the receptors and G-proteins in Sf9 cells, both 5HT2c and 5HT1A appeared to activate Gi significantly more than Gq. However, when receptors and G-proteins were expressed separately and examined by in vitro reconstitution, the 5HT2c displayed the expected preference for Gq, while 5HT1A preferred Gi. These experiments reveal a significant artifact of co-expression of GPCR and G-proteins in heterologous cells as a method to examine receptor signaling. Therefore, we have re-examined the structural determinants of GPCR-selectivity for chimeric G-protein alpha subunits composed of sequence from Gq and Gi proteins using in vitro assays. As opposed to earlier investigations using cell-based assays, our studies have identified the region of Gq which is selectively recognized by the 5HT2c as residues 301-322 and not the C-terminal 5 amino acids (350-353) as previously reported. In collaboration with Dr. Susan Sullivan, we have identified, cloned and expressed the entire repertoire of human bitter taste receptor (hT2R) genes. We have used the expressed hT2Rs to identify both the bitter compounds recognized by several of these receptors and the G-protein signaling pathways activated in response to bitter substances. Because the 25 hT2R genes must recognize a very large number of toxic substances, we had predicted a broad specificity with each of the hT2Rs recognizing a number of bitter compounds. While two of the h2TR genes encoding broadly tuned bitter receptors, surprisingly, two of the structures respond uniquely to a single compound of the some 60 tested. These investigations are providing fundamental insights into the molecular basis for human chemosensation. We have characterized a novel compound developed as a regulator of calcium homeostasis acting on the unique calcium sensing receptor (hCaR). Martial Ruat and colleagues from the CNRS, France, have provided the compound Calindol for our testing with transmembrane core domain (7TM) constructs we have produced from hCaR in examination of the fundamental mechanism for signaling by this unique receptor. We have established that Calindol acts both as a synergist for the natural calcium activation of the unmodified hCaR and as a potent agonist of the 7TM construct which lacks calcium ion sensing. Our data favor a new model for the molecular regulation of the hCaR which may pertain to the entire family of similar structures encoded in the human genome, including the receptors for the neurotransmitters GABBA and glutamate. Our data also provide an approach for studying the signaling properties of the more than 30 uncharacterized similar receptors identified in the human genome. Lastly, R. Victor Rebois has joined the laboratory and continued a project establishing resonance energy transfer-based methods for investigating the cellular localization and protein interactions of GPCRs, G-proteins and cellular effectors. We have initiated a research project to validate the protein-interactions of novel fluorescent-constructs for GPCRs and G-protein subunits using our in vitro biochemical and biophysical methods. Our initial study has focused on the D4 Dopamine receptor, as this GPCR cannot be labeled by the jellyfish fluorescent proteins.