Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution

Abstract

Agonist binding to G-protein-coupled receptors (GPCRs) triggers signal transduction cascades involving heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of conformational changes upon agonist binding, the details of interaction with the different G proteins, and the transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by coimmunoprecipitation the capability of 120 different G-protein combinations consisting of α i1 or α sL and all possible βγ-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein subunits to form heterotrimeric complexes, including βγ-dimers previously thought to be nonexistent, except for combinations containing β 5 . A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild type and a stabilized mutant indicated a preference for those Gα i1 βγ combinations containing γ 1 and γ 11 . Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes.heterotrimeric G proteins | G-protein-coupled receptor | membrane protein | protein complex | protein-protein interaction

Interaction of lipids with the neurotensin receptor 1

“…NTS1 is able to activate a range of signalling pathways through interaction with G q/11 at its intracellular loop 3 (IC3) [32], and through interaction with G i/o and G s at the first half of its C-terminal portion [33,34] and a recent study shows that this is complicated by the possible G αβγ heterotrimers that can interact with NTS1 [35]. Some transduction pathways can be suppressed by mutation or deletion of particular intracellular domains while leaving unrelated pathways intact, suggesting that pathways can be individually regulated by direct interactions with the receptor [36].…”

Association with the Plasma Membrane Is Sufficient for Potentiating Catalytic Activity of Regulators of G Protein Signaling (RGS) Proteins of the R7 Subfamily

“…G␣o). Lipid raft compartmentalization may further impact signaling due to G protein subunit diversity (16 G␣, 5 G␤, 12 G␥) providing hundreds of possible heterotrimeric combinations (45,46), each of which with a differing intrinsic lipid modification footprint (i.e. G␣o is palmitoylated and myristoylated whereas G␥1 is farnesylated) (47).…”

Hyperplasia in glands with hormone excess

“…The CaS-R on the parathyroid cell transduces hypercalcemia through Gq and/or Ga11, to activate phospholipase C, and thereby to raise inositol phosphates and to mobilize Ca++ from stores in the cytoplasm (Wettschureck N, Lee E, et al 2007; Conigrave AD, Ward DT 2013; Brown EM 2013; Hillenbrand M, Schori C, et al 2015; Cocco L, Follo MY, et al 2015; Nesbit MA, Hannan FM, et al 2013B ). Lowering of extracellular Ca++ or loss of function mutations of the CaS-R as in NSHPT stimulate secretion and hyperplasia in the parathyroid cells.…”