Reconstitution Reveals Additional Roles for N- and C-Terminal Domains of Gα in Muscarinic Receptor Coupling

Abstract

The molecular basis for selectivity of M1 and M2 muscarinic receptor coupling to heterotrimeric G proteins has been studied using receptors expressed in Sf9 cell membranes and reconstituted with purified chimeric G(alpha) subunits containing different regions of Gi1alpha and Gq(alpha). The abilities of G protein heterotrimers containing chimeric alpha subunits to stabilize the high-affinity state of the receptors for agonist and to undergo receptor stimulated guanine nucleotide exchange was compared with G protein heterotrimers containing either native Gi1alpha or Gq(alpha). The data confirm the importance of the proper context of the C-terminus of Galpha by demonstrating that the C-terminus of Gi1alpha, when placed in the context of Gq(alpha), prevents coupling to muscarinic M1 receptors, while the C-terminus of Gq(alpha), when placed in the context of Gi1alpha, prevents coupling to muscarinic M2 receptors. However, C-terminal amino acids of Gq(alpha) placed in the context of Gi1alpha were not sufficient to allow M1 receptor coupling, nor were C-terminal amino acids of Gi1alpha placed in the context of Gq(alpha) sufficient for M2 receptor coupling. The unique six amino acid N-terminal extension of Gq(alpha) when added to the N-terminus of Gi1alpha neither prevented M2 receptor coupling nor permitted M1 receptor coupling. A Gi1alpha-based chimera containing both N- and C-terminal regions of Gq(alpha) gained the ability to productively couple M1 receptors suggesting that the proper context of both N- and C-termini is required for muscarinic receptor coupling.

G Proteins in Drug Screening: From Analysis of Receptor-G Protein Specificity to Manipulation of GPCR-Mediated Signalling Pathways

“…(v) The serotonin 5-HT1B receptor couples to G protein heterotrimers containing either Gαi1, Gαi2, Gαi3, or Gαo but not Gαt although their Ctermini are highly homologous [71]. To date, at least 4 other regions within Gα have been shown to be involved in receptor interaction: The amino-terminal domain [21,[72][73][74], the α2 helix and α2-β4 loop regions [26,27], the α4 helix and α4-β6 loop domain [26,28,61,75,76], the α5 helix [77,78], the α3-β5 region [79], and a small segment within the loop linking the N-terminal α helix to the β1 strand of the ras-like domain [32]. Of those, the extreme N-and Ctermini, the αN-β1 loop, the α4-β6 region, and the α5 helix contribute to the specificity of Gα-receptor interaction ( Fig.…”

Recent advances in computational studies of GPCR-G protein interactions

“…In addition to structural biology, experimental techniques including mutagenesis Burstein, Spalding, & Brann, 1998;Chen et al, 2010;Conklin, Farfel, Lustig, Julius, & Bourne, 1993;Erlenbach et al, 2001;Kostenis, Conklin, & Wess, 1997;Liu, Conklin, Blin, Yun, & Wess, 1995;Marin, Krishna, & Sakmar, 2001;Moro, Lameh, Hogger, & Sadee, 1993;Preininger et al, 2009;Schoneberg, Kostenis, Liu, Gudermann, & Wess, 1998;Slessareva & Graber, 2003;Valiquette, Parent, Loisel, & Bouvier, 1995;Wacker et al, 2008;Xiao et al, 1999), nuclear magnetic resonance (NMR) (Kim et al, 2013), hydrogen-deuterium exchange mass spectrometry (HDXMS) (Chung et al, 2011;Orban et al, 2012), and double electron-electron resonance spectroscopy (DEER) (Van Eps et al, 2018) have been utilized to investigate the GPCR-G protein interactions Moreira, 2014;Preininger, Meiler, & Hamm, 2013). While the C-terminal α 5 helix in the G α subunit has been suggested as the primary driver for specific receptor recognition (Blin et al, 1995), the G α α N helix and receptor intracellular loop (ICL) 2 and transmembrane (TM) helix 6 further contribute to the GPCR-G protein coupling specificity (Burstein et al, 1998;Chen et al, 2010;Neumann, Krause, Claus, & Paschke, 2005;Preininger et al, 2013;Timossi et al, 2002;Zhou, Yan, Yamamoto, & Tai, 1999).…”

Estudos conformacionais de seqüências hidrofóbicas de domínios de receptores acoplados a proteínas G (GPCR) e da proteína G. Um estudo de CD e espectroscopia de fluorescência

“…Conforme discutido na seção 5.2.1, a parte distaI do C-terminal de G u é importante para o acoplamento com o receptor (138,(240)(241)(242). Além de Wess et aI.…”

“…Além de Wess et aI. (240), Graber et alo (242,243) também mostraram através de mutações que os últimos cinco resíduos do C-terminal de Ga são importantes para o acoplamento da proteína G com o receptor muscarínico. Essa região forma uma dobra (3, conforme discutido acima (seção 5.2.1).…”