Selective Inhibition of Heterotrimeric GsSignaling

Abstract

The blockade of heptahelical receptor coupling to heterotrimeric G proteins by the expression of peptides derived from G protein G␣ subunits represents a novel means of simultaneously inhibiting signals arising from multiple receptors that share a common G protein pool. Here we examined the mechanism of action and functional consequences of expression of an 83-amino acid polypeptide derived from the carboxyl terminus of G␣ s (GsCT). In membranes prepared from GsCT-expressing cells, the peptide blocked high affinity agonist binding to ␤ 2 adrenergic receptors (AR) and inhibited ␤ 2 AR-induced [35 S]GTP␥S loading of G␣ s . GsCT expression inhibited ␤ 2 AR-and dopamine D 1A receptor-mediated cAMP production, without affecting the cellular response to cholera toxin or forskolin, indicating that the peptide inhibited receptor-G s coupling without impairing G protein or adenylyl cyclase function. [35 S]GTP␥S loading of G␣ q/11 by ␣ 1B ARs and G␣ i by ␣ 2A ARs and G q/11 -or G i -mediated phosphatidylinositol hydrolysis was unaffected, indicating that the inhibitory effects of GsCT were selective for G s . We next employed the GsCT construct to examine the complex role of G s in regulation of the ERK mitogen-activated protein kinase cascade, where activation of the cAMP-dependent protein kinase (PKA) pathway reportedly produces both stimulatory and inhibitory effects on heptahelical receptor-mediated ERK activation. For the ␤ 2 AR in HEK-293 cells, where PKA activity is required for ERK activation, expression of GsCT caused a net inhibition of ERK activation. In contrast, ␣ 2A AR-mediated ERK activation in COS-7 cells was enhanced by GsCT expression, consistent with the relief of a downstream inhibitory effect of PKA. ERK activation by the G q/11 -coupled ␣ 1B AR was unaffected by GsCT. These findings suggest that peptide G protein inhibitors can provide insights into the complex interplay between G protein pools in cellular regulation.Heptahelical, or G protein-coupled, receptors represent the single most diverse class of cell surface receptors, both evolutionarily and within the human genome. The basic unit of G protein-coupled receptor signaling is composed of three parts as follows: a heptahelical receptor, a heterotrimeric G protein, 1 and an effector, such as a G protein-regulated enzyme or ion channel. The binding of an extracellular agonist ligand to the receptor changes its conformation so as to permit productive coupling with the G protein, thereby catalyzing the exchange of GTP for GDP on the G␣ subunit, and dissociation of G␣-GTP from G␤␥ subunits. Regulation of effectors is achieved through their interaction with free GTP-bound G␣ or G␤␥ subunits. Based upon data from crystallographic, biochemical, and mutagenesis studies, physical coupling of receptor and G protein is thought to involve primarily the second and third intracellular domains of the receptor, which make physical contact with the carboxyl terminus of the G␣ subunit (1-6). In particular, the last ϳ50 amino acids of the G␣ subunit are important ...

Essential roles of Gα12/13 signaling in distinct cell behaviors driving zebrafish convergence and extension gastrulation movements

“…The COOH-terminal region of G protein α subunits has been shown to be a major binding site of G proteins to their cognate receptors ( Hamm, 1998 ). Overexpression of CT-peptides in cells or mice has been shown to block competitively the receptor sites that normally bind to G proteins, leading to specific blockade of the respective Gα signaling ( Akhter et al, 1998 ; Gilchrist et al, 1999 , 2001 ; Feldman et al, 2002 ; Arai et al, 2003 ). Strikingly, compared with their control siblings, embryos injected with synthetic RNAs encoding the CT-peptides (the last 11 aa) of Gα 12 or Gα 13 (Gα 12 -CT, Gα 13 -CT) exhibited shortened body axes with broader notochords and somites ( Fig.…”

Role of LRP1 and ERK and cAMP Signaling Pathways in Lactoferrin-Induced Lipolysis in Mature Rat Adipocytes

“…For example, PKA inhibits c-Raf activity by phosphorylating c-Raf-Ser259 [ 49 , 50 ]. PKA phosphorylation of certain GPCRs switches their signaling from G s -stimulation of AC to G i -coupled activation of ERK [ 51 ]. Future studies will be required to investigate the biological consequences of the interaction between cAMP and ERK signaling pathways in lipolysis.…”

Signal Protein-Derived Peptides as Functional Probes and Regulators of Intracellular Signaling

“…The expression of 83-mer polypeptide derived from C-terminal region of the G α s induces the inhibition of β 2 -AR- and D 1A -DR-mediated cAMP production in HEK293 cells [146]. Short synthetic peptides corresponding to progressively longer segments of the G α s C-terminus, 384–394, 382–394, 380–394, 378–394(C 379 A), 376–394(C 379 A), and 374–394(C 379 A), stimulate specific binding of selective agonist CGS21680 to the G s -coupled A 2A -adenosine receptor in the rat striatal membranes both in the presence and in the absence of GTP γ S, a nonhydrolysable GTP analog, which uncouples G proteins from receptors (Table 1).…”