Signaling Specificity of the Gα i G Protein Subfamily8.5.5

Abstract

G protein-coupled receptors, or GPCRs, are a major class of cell surface receptors that transduce extracellular stimuli to intracellular signaling events via G proteins. GPCRs catalyze nucleotide exchange on the G protein Gα subunit leading to dissociation from Gβγ subunits. The canonical role of one family of G protein alpha subunits, Gαi, is binding and inhibition of the activity of adenylyl cyclase, leading to reduction in the level of 3′,5’-cyclic adenosine monophosphate (cAMP) in cells. Using proximity labeling mass spectrometry, our laboratory has recently discovered a novel role for Gαi in the activation of PDZ-RhoGEF (PRG), a guanine nucleotide exchange factor for the small G protein RhoA. Surprisingly, we found significant differences in the ability of Gαi1, Gαi2, and Gαi3 isoforms to activate PRG in a cell-based SRE-luciferase assay, despite over 85% sequence identity and equivalent inhibition of their common effector adenylyl cyclase. Gαi1 and Gαi3 strongly activate PRG, while Gαi2 shows very weak activation. A majority of the differences in these proteins are located in the helical domain of Gαi, which supports the central hypothesis that members of the Gαi subfamily differentially bind and activate PRG with specificity determined by structural features in the helical domain. To investigate this, the amino acid sequence of Gαi1 was divided into an N-terminal region, the helical domain (HD), and a C-terminal region. Protein chimeras were then generated, substituting one of each of these regions from Gαi1 into Gαi2 to determine if any of these domains are sufficient to confer the ability of the specific Gαi2-Gαi1 chimera to activate PRG in a SRE-luciferase assay in co-transfected HEK293 cells. All chimeras were validated for proper expression in HEK293 cells using western blotting, and all equally inhibited forskolin-stimulated cAMP production. The Gαi2-Gαi1-HD chimera activated PRG to levels similar to Gαi1, while the N-terminal and C-terminal chimeras did not activate PRG. This demonstrates that the helical domain of Gαi1 is sufficient for conferring the ability to activate PRG and structural features within this domain are responsible for the specificity amongst the Gαi1, 2, and 3 subtypes. In an effort to determine the structural features within the HD that confer Gαi-dependent activation of PRG, three subdivisions of the Gαi1 HD were then substituted into Gαi2. None of these subdivisions substituted alone resulted in activation of PRG, indicating that the requirements for PRG activation likely lie in three-dimensional coordination of multiple key residues throughout the helical domain of Gαi. Current and future work will focus on clusters of residue substitutions using three-dimensional structures and sequence alignments as a guide, substituting amino acids common between Gαi1 and Gαi3 but different in Gαi2. Constructs of interest will be examined in cell-based assays as well as purified and examined for in vitro binding to PRG. The resulting data have the potential to change the existing model for signal transfer from Gαi proteins to effectors and provides some of the first evidence for Gαi subtype-effector specificity. 8.5.5