Inactivation of the guanine nucleotide regulatory site mediating inhibition of the adenylate cyclase in hamster adipocytes

Abstract

The influence of N-ethylmaleimide and trypsin was studied on stimulatory and inhibitory regulations of the hamster adipocyte adenylate cyclase. Treatment of intact adipocytes or adipocyte ghosts with N-ethylmaleimide decreased basal and forskolin-stimulated adenylate cyclase activities. In the pretreated membrane preparations, inhibition of the enzyme by GTP and by stable GTP analogues was abolished. Concomitantly, activation of the adenylate cyclase by NaCl and its inhibition by the antilipolytic agents, prostaglandin E1 and nicotinic acid, were obliterated. In contrast, adenylate cyclase stimulation by ACTH and stable GTP analogues was not impaired but rather increased. Similarly, the NaCl-induced attenuation of the ACTH-stimulated enzyme activity was increased by the N-ethylmaleimide treatment. Limited proteolysis of hamster adipocyte ghosts with trypsin also obliterated GTP and prostaglandin E1-induced inhibitions and NaCl-induced activation of the adenylate cyclase. In contrast, adenylate cyclase activity stimulated by isoproterenol was increased after trypsin treatment. The data suggest that the activity of the adenylate cyclase is regulated via two distinct guanine nucleotide sites and that treatment with N-ethylmaleimide and limited proteolysis with trypsin functionally eliminates the regulatory site mediating adenylate cyclase inhibition, leading to a state where the enzyme activity is regulated only via the stimulatory site. The differential effects of these treatments on NaCl-induced activation and attenuation of the adenylate cyclase suggest that sodium acts on both regulatory sites in an inhibitory manner, and that by the functional elimination of the inhibitory site, only the sodium-induced attenuation of the adenylate cyclase via the stimulatory site is observed.

Islet‐activating protein prevents nicotinic acid‐induced GTPase stimulation and GTP but not GTPγS‐induced adenylate cyclase inhibition in rat adipocytes

“…Similar to that described for hamster adipocytes [9] and cyc-variants of S49 lymphoma cells 114,151, the rat adipocyte adenylate cyclase stimulated by forskolin was inhibited by the stable GTP analog, GTP+, much more potently than by GTP. Whereas GTPyS caused the same maximal degree of inhibition as observed with GTP, haIfmaximal inhibition was obtained with only 5 nM GTPyS.…”

“…Some data presented may also suggest that GTP hydrolysis is required for Ni-mediated adenylate cyclase'inhidition and IAP prevents GTP-dependent inhibition of adenylate cyclase by inhibition of Ni-associated GTPase stimulation. However, the data reported with stable GTP analogs in various cellular systems [8,9,14,15,18] and those shown herein with GTPyS in rat adipocytes are in apparent contradiction to this assumption. It may be speculated that stable GTP analogs not only act in a manner similar to GTP but that these analogs may have additional unknown effects on the adenylate cyclase system; e.g., causing conformational changes of the guanine nucleotide-binding coupling component(s).…”

“…There is increasing evidence that inhibition of adenylate cyclase by hormones and neurotransmitters is mediated by a guanine nucleotide-binding, regulatory component (Ni) that is distinct from that (N8) mediating adenylate cyclase stimulation by hormones and stable GTP analogs [17], Ni apparently also mediates adenylate cyclase inhibition by stable GTP analogs [9,18]. This assumption has been corroborated by findings in cyc-variants of S49 lymphoma cells.…”

“…imidodiphosphate and guanosine 5 ' -O-(3-thiotriphosphate) (GTP-S) [8,9]. Hormonal factors that inhibit adenylate cyclase stimulate a high affinity GTPase in adipocyte membranes, which is not affected by cholera toxin [lO,ll].…”

“…Adenylate cyclase activity was determined as in [9] with 50 /tM [~u-~~P]ATP (-0.4 &X/tube), 2 mM MgClz, 0.1 mM cyclic AMP, 1 mM 3-isobutyl-1-methylxanthine, 5mM creatine phosphate, 0.4 mg/ml creatine kinase, 2 mg/ml bovine serum albumin and 50 mM triethanolamine-HCl (pH 7.4) in 100 ~1 total vol. Reactions were initiated by the addition of adipocyte ghosts (5-30,ug protein/tube) and conducted for 10 min at 25°C.…”