The Role of Distal Helix 5 as a Determinant of GPCR‐G protein Coupling Selectivity

Abstract

G protein‐coupled receptors (GPCRs) interact with heterotrimeric G proteins to mediate important physiological responses, and compounds that bind to GPCRs represent approximately one third of FDA‐approved drugs. In humans, hundreds of GPCRs converge onto 16 G alpha subunits that are grouped into four families, and individual receptors select between these subtypes. Prior work suggests that the distal half of helix 5 (H5), the extreme C‐terminus of the G alpha subunit, is a key determinant of GPCR‐G protein coupling selectivity. The objective of this study was to determine the relative contributions of the extreme C‐terminus (H5.17‐H5.26) and the rest of the G alpha subunit (HN.1‐H5.16) to GPCR‐G protein coupling selectivity.We constructed a panel of G alpha subunits that included a wild‐type G protein from each of the four families (Gs‐long, Gi1, Gq, and G12), and all twelve possible chimeras where the last 10 amino acids (H5.17‐H5.26) of each G alpha subunit is replaced by those of another (e.g. Gsi‐10). These formed heterotrimers with venus‐labeled betagamma subunits, and coupling to Rluc8‐labeled GPCRs was measured using bioluminescence resonance energy transfer (BRET). BRET was measured in the presence and absence of both agonists and nucleotides in digitonin‐permeabilized HEK293 Gs/q/12 knockout cells which, together with pertussis toxin, were used to minimize interference from endogenous G proteins.Our results suggest that distal H5 influences GPCR‐G protein coupling, but by itself is not sufficient to determine selectivity. Furthermore, for some receptors, exchanging distal H5 had little effect on G protein coupling. For example, M3 muscarinic acetylcholine receptors coupled to Gqs‐10 more strongly than to Gsq‐10, suggesting that for this receptor the key selectivity determinants reside proximal to distal H5. In addition, the distal H5 of G12 was generally permissive, e.g. A1 adenosine receptors coupled to Gi12‐10 nearly as well as wild‐type Gi1. Taken together our results suggest that GPCR‐G protein selectivity determinants are likely to be distributed throughout the G protein alpha subunit, and distal H5 is only one such determinant. Moreover, G protein specificity determinants are likely to vary for different GPCRs. Future experiments will specifically address proposed specificity‐determining sites outside of distal H5.Support or Funding InformationSupported by NIH grants GM078319 and GM109879.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.