Proc. Natl. Acad. Sci. USA Vol. 76, No. 7, pp. 3189-3193, July 1979 Biochemistry Coupling of the glucagon receptor to adenylyl cyclase by GDP: Evidence for two levels of regulation of adenylyl cyclase* (rat liver plasma membranes/GTPase/guanosine 5'-[,'y-imidotriphosphate/adenylate cyclase) RAVI IYENGAR AND LUTZ BIRNBAUMFR Department of Cell Biology, Baylor College of Medicine, 1200 Moursund Avenue, Houston, Texas 77030 Communicated by Edwin G. Krebs, April 13,1979 ABSTRACT In rat liver plasma membranes preactivated clizing activity, for the analog appears to mimic the action of with guanosine 5'-[fl,y-imidojtriphosphate (GuoPP[NHI]), GDP GTP and, because it cannot be hydrolyzed by GTPase due to promoted coupling of occupied glucagon receptor to adenylyl the 3,-y-imido linkage, causes accumulation of the system as cyclase [adenylate cyclase; ATP, pyrophosphate-lyase (cyclizing), an enzyme-GuoPP[NH]P complex. Excess GDP, on the other EC 4.6.1.1] with an apparent association constant Ka of 0.1-0.15 hand, leads to a system with very low cyclizing activity (basal AM. The apparent Ka for the same effect of GTP was 0.2 ,M. The effect of GDP was shown not to be due to GTP formed by activity), for it appears that the GDP causes accumulation of putative transphosphorylation reaction(s) when ATP was present the system as an enzyme-GDP complex with low activity not in the assay as substrate. In membranes not preactivated with allowing the formation of enzyme-GTP and the ensuing acti- GuoPP[NH]P, GDP both competitively inhibited GuoPP[NH]P vation of the cyclization reaction. Evidence both from Levinson stimulation of adenylyl cyclase (Ki 0.10 AM) and supported and Blume (16) using neuroblastoma cells and more recently stimulation of cyclizing activity (apparent Ka 0.10 MM) by glu- from Cassel and Selinger (17) using turkey erythrocytes suggests cagon. These effects of GDP occurred in the absence of added that the affinity and the release rate of GDP from the system GTP and in the absence of sufficient formation of GTP by put- turn- ative transphosphorylation reaction(s) to account for them. It may be the limiting steps regulating the system's rate of is concluded that two levels of regulation of liver adenylyl cy- over-i.e., the rate at which, under the influence of GTP, the clase (cyclizing) activity must exit. One level is termed "receptor reaction sequence free enzyme enzyme-GTP - enzyme- regulation"; it depends on occupancy of a receptor-related R site GDP free enzyme occurs. Furthermore, work by the same by nucleotide and is specific for either GDP or GTP. The second authors suggests that receptor stimulation of cyclizing activity level of regulation is termed "GTPase regulation"; it is inhibited may be merely the result of a stimulation of the GDP release by GDP, depends on both GTP and GTPase and accounts for rate. Thus, Levinson and Blume (16) demonstrated that hor- activation of cyclizing activity by nonhydrolyzable analogs of neuroblastoma cell adenylyl cyclase de- GTP. The data suggest that both levels of regulation coexist and mone treatment of may synergize, one mediating responses to stimuli external to creases the affinity of the system for inhibitory GDP (tested the cell (receptor regulation) and the other mediating stimuli against GuoPP[NH]P), and Cassel and Selinger (17) showed of intracellular origin (GTPase regulation). that hormone treatment of turkey erythrocyte membranes preloaded with [3H]GDP results in rapid release of the label. Adenylyl cyclases [adenylate cyclase; ATP pyrophosphate-lyase Thus, the concept has emerged that positive coupling of hor- (cyclizing), EC 4.6.1.1] are complex enzyme systems that ap- mone-receptor complex to the adenylyl cyclase system (i.e., pear to be composed of two dissimilar types of subunits, each stimulation of cyclizing activity) involves the dissociation of having a separate catalytic activity: (i) a C subunit, or adenylyl GDP from the inactive state of the system followed by binding cyclase proper, bearing the catalytic site for the cyclizing re- of GTP and activation of ATP to cyclic AMP conversion (16, action and converting ATP to adenosine 3',5'-monophosphate 17). Therefore, GTP and its associated GTPase appear to play (cAMP) plus PPH, and (ii) a G subunit or GTPase bearing a an obligatory and central role in receptor regulation of the basic guanyl nucleotide binding site (G site) and catalyzing the hy- two-subunit adenylyl cyclase system. drolysis of GTP to GDP plus Pi (1-9). Optimal expression of It might then be envisioned that, in the absence of the GTP cyclizing activity has been shown to depend on the presence and GTPase-driven turnover of the system, hormonal regulation of and the interaction between both subunits (3, 6, 10). Ac- of enzymatic activity cannot occur. While this supposition cording to genetic (3, 6, 10), biochemical (2, 7-9), and kinetic appears to hold true in many instances in which the system is (11) studies, the enzyme system appears to exist in at least two fully activated either due to binding of a ligand not susceptible states of cyclizing (and possibly also GTPase) activity: one with to hydrolysis by GTPase [e.g., GuoPP[NH]P orGTP[S] (4, 5, 11, very little activity (basal activity) and the other active, the active 12, 15, 18, 19)], or due to the inhibition of the GTPase itself [e.g., to presence of ADP-ribosylation by cholera toxin (6-8), or intracellular factors state(s) being induced (stabilized) by GTP. Due (2.0)], experiments testing for the effects of GDP on hormonal the GTPase, the basic adenylyl cyclase system can be assumed stimulation of the glucagon-sensitive adenylyl cyclase system to be in constant turnover (12). indicate otherwise. The present report presents some of our The G site, as its name indicates, is specific for guanyl nu- experiments on the capacity of GDP to promote positive cou- cleotides. It interacts not only with GTP but also with GDP and pling of glucagon receptor complex to the adenylyl cyclase GTP analogs such as guanosine 5'-[f3,y-imido]triphosphate system in the absence of measurably significant concentrations (GuoPP[NH]P) (13) and guanosine 5'-[y-thio]triphosphate of GTP. (GTP[S]) (14, 15). In the presence of either [GuoPP[NH]P or in two differing GDP, the system's turnover cycle is blocked Abbreviations: GuoPP[NHIP, guanosine 5'-[3,,y-imido]triphosphate; states of activity. Thus, GuoPP[NH]P leads to enhanced cy- AdoPP[NHIP, adenosine 5'-[3,'y-imido]triphosphate; GTP[S], gua- nosine 5'-[-y-thioltriphosphate; cAMP, adenosine 3',5'-monophos- The publication costs of this article were defrayed in part by page phate. charge payment. This article must therefore be hereby marked "ad- * This is the second paper in a series exploring the characteristics of vertisement" in accordance with 18 U. S. C. §1734 solely to indicate hormone receptor coupling to adenylyl cyclase. The first paper is ref. this fact. 22. 3189 Downloaded by guest on September 28, 2021 3190 Biochemistry: Iyengar and Birnbaumer Proc. Natl. Acad. Sci. USA 76 (1979) EXPERIMENTAL PROCEDURES evaluated by seeding small aliquots (1-2 Ml) of the stopped re- Materials. [a-32P]GTP and [a-32P]ATP were purchased action mixtures on plastic-covered thin-layer sheets of poly- from International Chemical and Nuclear Corporation or ethyleneimine cellulose, chromatographing (ascending) with prepared in our laboratory as described (21). [a-32P]GDP was 1 M LiCl as developing solvent, and determining the distribu- prepared by acid hydrolysis (pH 2) of [a-32P]GTP. The [a- tion of radioactivity by liquid scintillation counting. 32P]GDP was separated from [32P]GMP and [a-32P]GTP by Protein was determined by the Lowry method (28) with chromatography on DEAE-Sephadex (21). Unlabeled GDP was bovine serum albumin as standard. obtained from Sigma (catalog number 6506) and was found to contain less than 1% GTP as assessed by chromatography on RESULTS DEAE-Sephadex (21). Plastic-backed polyethyleneimine-cel- Experiments from several laboratories (28, 29, 19), including lulose thin-layer sheets were from Brinkmann. The sources of our own (30, 22), indicate that two separate nucleotide sites are all other materials used throughout are those described (22, involved in hormonal stimulation of adenylyl cyclases such as 23). the glucagon-sensitive one from liver membranes. One site is Adenylyl Cyclase Assay. Conditions of assay included [a- the G site on the G subunit of the basic system referred to in the 32P]ATP (50-100 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels), 0.5 introduction. The second site is a receptor-related site (R site) mM ATP or 0.5 mM adenosine 5'-[3,,y-imido]triphosphate responsible for (AdoPP[NH]P), 5.0 mM MgCl2, 1.0 mM EDTA, 1.0 mM regulation of receptor behavior-i.e., binding [3H]cAMP (approximately 10,000 cpm per assay), 25 mM affinity (31), binding rates (31), and coupling (22).t Because 1,3-bis[tris(hydroxymethyl)methylamino]propane-HCI (Sigma) the original work on nucleotide effects on binding of glucagon at pH 7.5, and partially purified liver plasma membranes (24) to liver membranes had shown GDP to be as good a ligand as at 0.01-0.03 mg of protein per ml-i.e., 0.5-1.5 Atg of mem- GTP in promoting dissociation of 125I-labeled glucagon from brane protein per 5S0-l assay. Incubations were for 5 min at its specific binding sites under conditions in which no tran- 32.50C, after which time they were stopped (22) and [32P]cAMP sphosphorylation was possible, it became of interest to deter- formed was assayed according to Salomon et al.