Stimulatory GTP regulatory unit Ns and the catalytic unit of adenylate cyclase are tightly associated: mechanistic consequences.

Abstract

Turkey erythrocyte membranes were solubilized in the mild detergent octylpenta(oxyethylene) [CH3(CH2)7-(OCH2CH2)50H], which possesses a high critical micelle concentration (=6 mM) and forms small, dynamic micelles. Both the native enzyme N,(GDP)-C and the p [NH] Receptors for hormones and neurotransmitters interact with an effector system within the plane of the membrane to generate the primary biochemical response. A major class of receptors consists of those that activate adenylate cyclase upon agonist binding (refs. 1 and 2; ref. 3 and references therein). The activation of the catalytic unit of adenylate cyclase depends not only on the occupancy of the receptor by an agonist but also on the simultaneous occupancy of the GTP regulatory protein Ns by GTP (4). A number of mechanisms have been proposed for the sequence of events leading from the step of agonist occupancy on the receptor to the production of cAMP from ATP by the catalytic unit C. Currently three types of mechanisms are being discussed (Fig. 1).Although there is considerable evidence that hormone activation of adenylate cyclase by 8-agonists (5-7), glucagon (8, 9), and neurohypophyseal hormone (10) occurs by the "collision coupling" mechanism, counter arguments have been raised. These arguments are based on two types of observations: (i) guanyl nucleotides reduce the affinity of fadrenergic receptors towards their agonists (11) and, therefore, Ns and R are associated (12), and (ii) the n-receptor in the presence of guanyl-5'-yl-imidodiphosphate (p[NH]ppG) can activate Ns in the total absence of C (13). These observations were taken to mean (12, 13) that Ns "shuttles" between R and C (Fig. 1).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. other at all times. This model predicts (i) linear dependence of the rate constant of adenylate cyclase activation on the concentration of receptor R; (ii) the interaction between N, and C is never rate limiting; (iii) noncooperative binding of H. Model 3: N, shuttle. In this model the receptor R, the GTP stimulatory unit N,, and the catalytic unit C are all separate and interact sequentially as depicted. This model predicts noncooperative binding of H. if HR-N'(GTP) does not accumulate, and complex kinetics of adenylate cyclase activation. In the extreme case in which one of the components is in great excess, the kinetics will approach first-order kinetics.In fact, these two observations do not contradict the collision coupling theory. First, although the f3-adrenergic receptor forms a stable complex with Ns, in the presence of a f3-agonist and in the absence of guanyl nucleotide, it readily decomposes upon GTP or p[NH]ppG binding (12). Second, the fact that R and Ns interact with each other in the absence of C does not disprove that in the native situation Ns and C always function as a complex. Indeed, to separate Ns from C, a combination of ...

Seven Transmembrane Receptor Core Signaling Complexes Are Assembled Prior to Plasma Membrane Trafficking

“…Most early evidence for existence of stable protein/protein interactions between G proteins and effectors came from in vitro biochemical studies (for example see Ref. 55 and for review see Ref. 56).…”

Heterotrimeric G proteins form stable complexes with adenylyl cyclase and Kir3.1 channels in living cells

“…Most of the evidence for the existence of stable protein-protein interactions between G proteins and effectors in mammalian cells comes from in vitro biochemical studies (e.g. Arad et al, 1984) (reviewed by Rebois et al, 1997). The first proposal that G proteins and effectors existed as a complex in cell membranes was based on data indicating that signal transduction displays first order kinetics (Levitzki, 1981;Levitzki, 1984;Levitzki, 1986;Levitzki, 1988a;Levitzki, 1988b;Marbach et al, 1990;Tolkovsky et al, 1982;Tolkovsky and Levitzky, 1981).…”

The role of G proteins in transmembrane signalling

“…Attachment of the a subunit to the cytoplasmic face of the membrane may be mediated by the fly complex (Sternweis, 1986) or by fatty acids covalently linked to the N-termini of some a subunits (Buss et al, 1987;Lochrie & Simon, 1988;Mumby et al, 1990). For a., which lacks covalently bound lipid, the effector itself, adenylate cyclase, may also provide an anchor to the plasma membrane (Arad et al, 1984; Levitzki, 1987). The nature of the attachment is important because G protein activation almost certainly involves dissociation of the a from the fly subunits, and if the latter provide the only membrane attachment the active a subunit would leave the membrane.…”

“…Under physiological conditions, it seems more likely that there is no significant dissociation of active a. subunits from the membrane. More direct evidence comes from the work of Levitzki and his colleagues who have consistently argued from both kinetic analyses (Tolkovsky & Levitzki, 1978a;Levitzki, 1986) and the physical association of G. and adenylate cyclase during substantial purification (Arad et al, 1984) that G., at least, remains tightly bound to its effector, and therefore to the membrane, throughout the G protein cycle. Even transducin, the G protein that is least tightly associated with the membrane, may not significantly dissociate from the disc membrane after activation under physiological conditions (Liebman et al, 1987;Uhl et al, 1990), although Chabre (1987) has argued that the°Ct-GTP subunit is soluble and becomes a cytoplasmic messenger.…”