Identification of receptor contact site involved in receptor–G protein coupling

Abstract

The mammalian G proteins transduce information from extracellular signals, including neurotransmitters, hormones and sensory stimuli, into regulation of effector enzymes or ion channels within cells. Triggered by appropriate extracellular signals, receptor proteins specifically activate members of the G protein family by catalysing replacement of GDP by GTP at the guanine nucleotide binding site. Like the receptor proteins, the heterotrimeric G proteins exhibit impressive structural similarities, suggesting that all receptor-G protein interactions use homologous structural elements and a single molecular mechanism. Topologically equivalent portions of each G protein may therefore interact with the appropriate receptor. We recently predicted the secondary structure of a composite G protein alpha-chain and proposed that a predicted amphipathic alpha-helix at the extreme carboxy-terminus of the polypeptide directly contacts receptors. This proposal has now been confirmed by sequencing complementary DNAs of the gene that encodes the alpha-chain (alpha s) of the stimulatory regulator (Gs) of adenylyl cyclase in wild-type cells and in a mutant mouse S49 lymphoma cell line, unc, in which Gs cannot be activated by hormone receptors. The sequences reveal a point mutation in the unc gene that substitutes a proline residue for an arginine near the carboxy-terminus of the alpha s-polypeptide. Expression of recombinant alpha s-unc in genetically alpha s-deficient S49 cells reproduces the unc phenotype.

Mutations at the Domain Interface of Gsα Impair Receptor-mediated Activation by Altering Receptor and Guanine Nucleotide Binding

“…Preparation of Stable Cell Lines-␣ s constructs were subcloned as HindIII fragments into the retroviral vector pMV7 (20) and then stably expressed as described (21), in a subclone of cyc Ϫ S49 lymphoma cells, cyc Ϫ kin Ϫ (22), in which cAMP-dependent protein kinase is inactivated. Single colonies containing the pMV7 vector were obtained using limiting dilution in microtiter wells and selection in G418 (1 mg/ml).…”

“…Of the mutated ␣ s residues, only Lys 305 and Tyr 311 are close to the interface in the structure of ␣ s ⅐GTP␥S (32). Although interactions between residues in switch III and the ␣D/␣E loop are important for receptor-mediated activation, the known receptor binding sites of ␣ s , the carboxyl terminus of ␣5 (13,21,34) and possibly the ␣4/␤6 loop (34), are not near this interface. ␣ subunits bind to ␤␥, which is required for receptormediated activation, via switches I and II and the amino terminus (10,11), which are also not near this interface.…”

A comparison of post‐receptor signal transduction events in Jurkat cells transfected with either IL‐8R1 or IL‐8R2 Chemokine mediated activation of p42/p44 MAP‐kinase (ERK‐2)

“…Saturation binding studies were subsequently performed in the presence and absence of PTx in order to test whether ADP-ribosylation of the G-protein interfered with receptor-ligand interactions. Table 1 shows that binding of ~25I-IL-8 to both receptors is unaltered by PTx treatment, suggesting that although PTx ribosylation leads to uncoupling of the G~-subunit [32,33], the interaction of IL-8 with its receptor binding site remains unaffected.…”

Studies of the regulation and function of the Gsα gene Gnas using gene targeting technology

“…Such somatic mutations have been identified in sporadic endocrine tumors (pituitary, thyroid, adrenal), fibrous dysplasia of bone, and the McCune-Albright syndrome (Lyons et al, 1990;Weinstein, 2002. The amino terminus and switch 2 regions interact directly with β-subunit to form the heterotrimer (Lambright et al, 1996;Wall et al, 1995), while the carboxyl terminus is important for receptor interactions (Conklin et al, 1996;Grishina & Berlot, 2000;Mazzoni et al, 2000;Schwindinger et al, 1994;Simonds et al, 1989;Sullivan et al, 1987). cAMP, the major second messenger resulting from G s α signaling, mediates its effects through direct binding and stimulation of several molecules, including protein kinase A (PKA), cAMPregulated guanine nucleotide exchange factors (cAMP-GEFs) (de Rooij et al, 1998;Kawasaki et al, 1998), and ion channels (Sudlow et al, 1993;Wainger et al, 2001).…”