Presenting Author University Antwerp Antwerpen, Belgium
G protein-coupled receptors (GPCRs) are successful drug targets due to their functional involvement in nearly every biological process. GPCRs can generate functional signals via both G proteins and β-arrestins. Our research has demonstrated that an additional non-G protein signaling function may exist for GPCRs, i.e. the GIT2 (GRK interacting transcript 2) signaling pathway. The structural bases of differential signaling paradigm control in GPCRs are still relatively unclear. Through structural modification of conserved structural motifs in GPCRs, we have sought to unravel this conundrum. The hydrogen-bond network, including transmembrane helix 1 (TM1) Asparagine (N1.50), TM2 Aspartate (D2.50), and TM7 Asparagine (N7.49), is conserved across the majority of Rhodopsin-like GPCRs and is known to affect GPCR signal initiation. Human GPR19 however possesses a natural variation in this network, i.e. a Lysine substitution for the TM7 Asparagine N7.49. Here we have introduced selective point mutations to several of these conserved sites and have investigated how these changes affect G protein, β-arrestin, and GIT2 signaling bias effects. We have investigated both the proteomic cellular response to expression level variation of wild type (WT) and mutant (MT) GPR19 forms, as well as the physical interactome of WT and MT forms by using quantitative mass spectrometry. With these data we have performed targeted bioinformatic analyses to investigate signaling bias effects at GPR19. Our results have demonstrated that the TM2 MT D2.50A was able to indeed shift signaling bias from β-arrestin paradigms to GIT2 signaling paradigms. Our research, as well as others, have shown that β-arrestin and GIT2 signaling can act in an opposite manner regarding stress resistance and DNA damage (both drivers of aging and age-related disease), with the former facilitating damage and the latter attenuating damage. Controlling the ability of a GPCR to selectively regulate these pathways could show beneficial effects concerning DNA damage in the context of aging and age-related disease.
This research was funded by the University of Antwerp Seal of Excellence Grant (41/FA010100/FFB210357), FWO-OP/Odysseus program (42/FA010100/32/6484) and the FWO PhD grant fundamental research (lt;agt;1198020Nlt;/agt;).
The hydrogen-bond network in canonical rhodopsin-like receptors includes the residues N1.50, D2.50 and N7.49. In the receptor GPR19 we mutated both sites, D2.50 and K7.49 to Alanine and investigated the proteome for the WT and the mutants with mass spectrometry. This data was used to determine enriched pathways and to determine the degree between G protein, β-arrestin and GIT2 signaling paradigms. The D2.50A mutant showed a shift from GIT2 to β-arrestin signaling paradigms compared to the WT.
