Multisite contacts involved in coupling of the β‐adrenergic receptor with the stimulatory guanine‐nucleotide‐binding regulatory protein

Abstract

Synthetic peptides, 12 -22 amino acid residues long, comprising the presumed coupling sites of the p-adrenergic receptor with the stimulatory guanine-nucleotide-binding regulatory protein (G,), were examined for their ability to modulate G, activation in turkey erythrocyte membranes. Three peptides corresponding to the second cytoplasmic loop, the N-terminal region of the third cytoplasmic loop, and the N-terminal region of the putative fourth cytoplasmic loop, compete synergistically with the hormone-stimulated receptor for G, activation with median effector concentrations of 15 -35 pM, or 3 -4 FM for combinations of two peptides. One peptide, corresponding to the C-terminal region of the third cytoplasmic loop, carries the unique ability to activate the G,-adenylatecyclase complex independent of the signalling state of the receptor. These observations are consistent with a dynamic model of receptor-mediated G-protein activation in membranes, where domains composed of the second, third and fourth intracellular loop of the receptor bind to and are interactive with the G-protein heterotrimer, resulting in ligand-induced conformational changes of the receptor. In response to hormone binding, the extent or the number of sites involved in interaction with G, may be readjusted using a fourth site. Modulation of coupling sites may elicit congruent conformational changes within the G, heterotrimer, with qualitatively different effects on GTP/GDP exchange in the a subunit of G, and downstream effector regulation. This model corroborates and expands a similar model suggested for activated rhodopsin-transducin interaction [Konig,

G-Protein binding domains of the angiotensin II AT1A receptors mapped with synthetic peptides selected from the receptor sequence

“…Our observation that three different competing peptides affect AT "A R-G-protein coupling is consistent with the hypothesis that multiple regions of the receptor form a three-dimensional binding surface to which the G-protein is coupled [8,14,28]. In the AT "A R, this binding surface comprises at least regions from i2, i3 and the C-terminal tail.…”

“…This juxtamembranous domain has a highly charged lysine-rich sequence, which, like the N-terminal region of i2, is predicted to form an amphiphilic helix. In fact, CD and NMR analysis of AT "A R peptide 300-320 has shown that this peptide, which is very similar to the one used in the present study, does form an amphipathic α-helix [27] and suggests that the helix-to-coil transition in i o is a mechanism by which G-proteins associate or dissociate from the AT "A R. In β-adrenergic receptors, peptide competition studies have shown that a similar lysine-rich amphiphilic helix in the proximal C-terminal tail plays a critical role in receptor-G s -protein coupling [8]. Palmitoylation of cysteine-322 in the C-terminal tail of the β-adrenergic receptors is speculated to form a short intracellular loop by embedding the palmitoyl moiety in the membrane.…”

“…Competing peptide experiments have implicated several regions of various receptors in G-protein coupling. The N-terminal regions of i2 and i3 and the proximal region of the cytoplasmic tail were implicated in agonist-mediated G-protein activation by rhodopsin and avian β-adrenergic receptors [8,11]. In α #A -adrenergic receptors, peptides derived from i2 and the C-terminal region of i3 abolished the receptor-G-protein interaction [10].…”

“…It is noteworthy that the DRY (Asp-ArgTyr) sequence, which appears in this segment, is highly conserved in the N-terminal region of i2 among members of the G-proteincoupled receptor superfamily [14]. In β-adrenergic receptors, a synthetic peptide from the N-terminal region of i2, including the DRY sequence, markedly attenuated agonist-induced stimulation of adenylate cyclase [8]. Mutation of the negativelycharged asparagine residue in the DRY sequence has been shown to reduce or abolish receptor-G-protein coupling in the AT "A R [13] as well as in the m " muscarinic, the β # -adrenergic and the α #A -adrenergic receptors [21][22][23].…”

“…Recent studies have shown that the AT "A R interacts with the pertussis-insensitive G q class of Gproteins [2], as well as the G "#/"$ family [3], and that the activated α subunit of the G-proteins, in turn, stimulates phospholipase-C β " [4,5]. The structure-function relationships between receptors and Gproteins have been elucidated for some G-protein-coupled receptors such as the β-adrenergic [6][7][8], α " -adrenergic [9] or α #A -adrenergic [10] receptors, rhodopsin [11] and the m # muscarinic receptors [12], using deletion and site-directed mutagenesis studies, as well as competition studies with synthetic peptides corresponding to the amino-acid sequences of the intracellular loops. These studies indicated that multiple domains located in the second (i2) and third (i3) intracellular loops and the Cterminal tail may play a role in the receptor-G-protein interaction in those receptors.…”