Regulation of Glucagon Receptor Binding - Semantic Scholar

Vol. 260,No. 13, Issue of July 5,pp. 7829-7835,1985 Printed in U.S.A.

THEJOURNAL

OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Regulation of Glucagon Receptor Binding LACK OF EFFECT OF MgAND

PREFERENTIAL ROLE FOR GDP* (Received for publication, September 12, 1984)

Francisco J. RojasS and LutzBirnbaumerj From the Department of CeU Biology, Baylor College of Medicine, Houston, Texas 77030

The effects of Mg2+and guanine nucleotides on glu- Studies carried out largely with the P-adrenergic receptor, but cagon binding to its receptor were studied using [1261- to some extent also with other receptors, have shown that the Tyr’o]monoiodoglucagon. Contrary to findings with B- receptor-N, interaction is under the regulation of hormones adrenergic receptors,high affinity bindingof the stim- or agonists, which bind to the receptor, and guanine nucleoulatory hormone was not dependent on M e and low tides and M e , which bind to N.. The effects of guanine affinity bindingcould be obtained on nucleotide addi- nucleotides and Mg2+ on agonist binding have been well tion regardless of presence of M8+. GDP, guanyl-5’- described and so have the activation ofN. byM$+ and yl thiophosphate (GDPBS), GTP, and guanyl-5’-yl im- guanine nucleotides and the stimulation of the activation idodiphosphate (GMP-P (NH)P) wereall able to induce process by agonist-receptor complexes. (For review see Ref. low affinity hormone binding. Since theN. component 1.) These studies led to the current recognition that both of adenylyl cyclase, with which the receptor interacts, receptor and N, exist in at least two forms or states: receptors is inactive in stimulating the catalytic component C of adenylyl cyclase in the absence of M e + , both before in Hand L states, exhibiting high and low affinity for agonists and active (Ns(a)) states,of and after GDP addition, it is suggested that N. has at (2, 3), and N. in inactive (NSco) which the active state is able to enhance the catalytic activity least two domains that change conformation independently of each other:a r domain, that interactswith the of the catalytic component C of the adenylyl cyclase system receptor and confers to it high affinity binding, and a while the inactive state is not (4-6).* Correlation of intrinsic c domain, that interacts with the catalyst C and stim- activity of a large number of catecholamine analogs having ulates it. It is suggested further that N. is r+c- when varying agonistic properties with their ability to stabilize the stabilizing the receptor in its conformation with high P-adrenergic receptor in its H state revealed that agonism is affinity for hormone, and r-c- when under the influ- proportional to the proportion of receptors that can be induced to adopt the H state as well as to the difference in ence of GDP which results in the receptor adopting the conformation that exhibits low affinity for the horaffinities between the H and L states (2, 3). The notion mone. Comparison of potencies of the fournucleotides emerged that the H state represents the active form of the to induce low affinity binding showed that GDP and receptor, i.e. the form that is required for facilitation of N. GDP@Swere equipotentand 10 times more potent than activation by guanine nucleotides and Mg. Biochemical and GTP and 100 times more potent than GMP-P(NH)P. kinetic studies suggested further that toadopt its H state the Under theconditions used it was impossible to substan- receptor requires the presence of Mg2f ( 7 , 8 ) and the inactive tiate that the effects of GTP or GMP-P(NH)P were not form ofN., i.e. the form that is found when guanine nucleodue to formation of GDP from GTP or presence of tides are absent (9). Thus, H type binding appears to be the GDP-like material in GMP-P(NH)P. It is suggested that, contraryto widely held opinions, GDP and GDP- binding property of the RN. complex in the presence of M$+. like compounds, and not GTP or its analogs, are re- Addition of GTP, or one of its non-hydrolyzable analogs, to sponsible for the lowering of the affinity of adenylyl a HRN,M$+ complex results in two consequences: (a) acticyclase stimulating receptors for their hormones or vation ofN. (4, 10) and ( b ) a change of the receptor conforagonists. Furthermore, the experiments suggest that mation to its L state, which is thought to be correlated with the c+ conformation of the c domain of N. co-exists with the dissociation of the receptor from the activating N protein and therefore to represent affinity of the free receptor (9,11). the r+and not the r- conformation of its r domain. Since N, is a GTPase (12,13), and since continuous hormonal stimulation of adenylyl cyclases is associated with increased GTP hydrolysis (14, 15), a turnover cycle has been proposed Adenylyl cyclase-stimulating receptors interact with Ne.’ to explain hormonal stimulation of N.: 1) R ( L state) combines with N. (inactive) to give RIL)N,(;),2) hormone (H) and M e * This work wassupported by National Institutes of Health Grants bind to the RIL)N.(i)complex to give HRImNsci,M$+(the soAM-19318, AM-26905, and AM-27682. The costs of publication of called “ternary complex” of DeLean et al. (2)), 3) GTP comthis article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Present address: Department of Obstetrics and Gynecology, University of Texas Medical School a t San Antonio, 7703 Floyd Curl Drive, San Antonio, T X 78284. § To whom correspondence should be addressed. The abbreviations used are: N., stiinulatory N; AMP-P(NH)P, adenyl-5”yl imidodiphosphate; BSA, bovine serum albumin; GMPPNH2, guanyl-5’-yl imidophosphate; GDPDS, guanyl-5’-yl thiophosphate; GMP-P(NH)P, guanyl-5”yl imidodiphosphate, C, catalytic

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unit of adenylyl cyclase; N, guanine nucleotide-binding regulatory stimulatory component of adenylyl cyclase; R, hormone receptor; 9, R RS, nucleoside triphosphate regenerating system; DTT, dithiothreitol; H or H state, stateof receptor with high affinity for hormone; L or L state, stateof receptor with low affinity for receptor. The active form of N. may merely be a different conformation of the N protein or, as suggested by studies with non-hydrolyzable analogs, represent the 01 subunit complexes with the guanine nucleotide unseparated from the 07 portion of the protein (44-47).

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Regulation of Glucagon Binding Receptor

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Liver Membranes plus bines with this complex and leads to formation of HR(L)plus 0.3nM Monoiodoglucagon (32.5OC) N.(,)Mg2+GTP, 4) the N protein hydrolyzes GTP with concomitant deactivation and subsequent release of GDP andMg Qlucagonto give N,ci), 5 ) the system re-initiates the cycle. A variant to 40-60 80-90 (% of max) stimulated: this cycle has been proposed whereby GDP dissociation does Ad. Cycl. Act. not occur until hormone and Mg2+ interact with the system (16, 17). Some data, however, are notexplained by the above scheme. For one, the potencies of nucleotides to activate N. do not correlate with those affecting ligand binding; for another, GDP appears to exert the same effect as GTP. Thus, Stadel and Lefkowitz (18) reported that GMP-P(NH)P is significantly less potent in affecting agonist binding than GTP. However, in all activity studies reported thus far (c.f Refs. 19 and 20) including that of Stadel and Lefkowitz (18), GMP(NH)P is either equally or up to 10-fold more potent than GTP in activating N., regardless of whether measured in the absence of hormone or in its presence. Furthermore, while cholera toxin treatment of membranes has eitherno effect or a slight potentiating effect on guanine nucleotide mediated Nucleotide Added (@I) activation of N, (19, ZO), it significantly decreases the potency of GTP and GMP-(NH)P toaffect agonist binding (18). That FIG. 5. Comparison of the potencies with which GDP, GTP, GDP addition leads to changes in hormone binding was first and GMP-P(NH)P affect [’aaI]monoiodoglucagon binding to reported by Rodbell et al. (21) and laterconfirmed in avariety liver membranes. Experimental conditions were identical to those of receptor systems including P-adrenergic receptors of S49 described in the legend to Fig. 4 with 5 mM M&lz present. Incubations testing for effects of GTP contained the nucleoside triphosphate cells and turkey erythrocytes(22). Its effect, therefore, seems regenerating system; those testing for effects of GDP and GMPto be as “universal” as that of GTP and its analogs. Since P(NH)P, did not. Specific binding of [‘z51]monoiodoglucagonwas levels of guanine nucleotides in intact cells are between 100 5700 f 350 cpm in the absence of the regenerating system and 5230 f 197 cpm in its presence (means f S.D. of triplicates). Nonspecific , l/lOth those of the corresponding adenine and 500 p ~ i.e. nucleotides, and since these are concentrations that appear binding determinedin quadruplicate in the presence of 1 pM of to be saturating with respect to N. (22), the question arises glucagon was unaffected by the presence of the regenerating system and averaged 1560 -t 89 cpm (mean f S.D. of sextuplicates). Vertical as towhen M e and agonists would find the R(L)N.(i)complex lines and numbers next to them indicate concentrations at which free of guanine nucleotides so that the activating R(m state nucleotides exert 50% of maximum effects. Shaded areas, concentracan be formed. One possibility is that the potency of GDP tions a t which these nucleotides exert 40-50% and 80-90% of their (i.e. its affinity for N,) is so low that it dissociates from N. effect to enhance glucagon-stimulated adenylyl cyclase activity under immediately after its formation. Activity studies, in which identical assay conditions of liver membrane, [12SI]monoiodoglucagon, MgClZ, and AMP-P(NH)P concentration and of temperature and GDP shows high potency in blocking stimulatory effects of time of incubation. For the restof the conditions, see Legends to Fig. GTP or GMP-P(NH)P, do not suggest this to be the case 4, text, and “Experimental Procedures.” (22). But, studies on the potency of GDP on agonist binding have not been reported and need to be determined. On investigating the regulation of hormone binding to the The present workwas undertaken to test, in a receptor glucagon receptor we discovered that in contrast to what is system other than the p-adrenergic system, which of the observed in the p-adrenergic receptor system, the glucagon regulatory features described for &adrenergic receptors are receptor does not require presence ofMgZ+ to exhibit high indeed of a “universal” character, what the potency of GDP affinity binding (Figs. 1, 2, 4, and 6). This allowed US to might be in affecting hormone binding, and whether the above investigate the order of potency with which guanine nucleomentioned discrepancies between potencies of nucleotides in tides cause the transition of high affinity binding to low acting on hormone binding versus acting on N, activation affinity binding, a phenomenon known to be N. mediated. apply to other systems as well. We have recently perfected a Experiments such asshown in Fig. 5, yielded EC50 values that method for the preparation of [‘251-Tyr’0]m~n~iodoglucag~n ranged between 0.05 and 0.09 p~ for GDP, 0.6 and 1.0 PM for for the study of glucagon receptor properties (23). We there- GTP, and 7 and 12 p~ for GMP-P(NH)P. In other experifore setout to study the regulation of its H to L state ments the potency of GDPPS ranged from 0.05 to 0.10 p M transitions under the influence of M$+, guanine nucleotides, (not shown). and glucagon. We found that M$+ is not necessary for the stabilization of the glucagon receptor in its H state and that DISCUSSION GDP seems to be the nucleotide preferred byN. to promote We showed that M$+ is not always required for observing states of N., the one the H to L transition.Sinceboth the H state of a receptor that couples to N. and that, of the stabilizing receptor in its H form and the one stabilizing the four nucleotides tested, GDP and itsanalog GDPSS are much receptor in its L form (or dissociating from it) are inactive, this defines the existence of at least two different inactive forms of the N p r ~ t e i n . ~ EXPERIMENTALPROCEDURES

ANDRESULTS4

3 A preliminary account of the findings wasgiven at the 43rd Annual Meeting of the American Diabetes Association, June 12-14, 1983, San Antonio, Texas (in Diabetes 32,Suppl. 1,page 44A, Abstr. 175). Portions of this paper (including “Experimental Procedures,” part

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of “Results,” and Figs. 1-4 and 6-9) are presented in miniprintat the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 84-M2857, cite the authors, and include a check or money order for $8.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

Regulation of Glucagon Receptor Binding more effective in promoting the H to L transition of receptors than GTP and GMP-P(NH)P. We feel that the lack of observing an effect of M e on glucagon binding is notdue to an artifactfor we saw no effect of M$+ on any of the parameters that can be observed in a hormone binding study of this type, including affinity and extent of binding of the receptor probe, capacity of the native hormone to displace the receptor probe in the absence or in the presence of guanine nucleotides, and apparent potencies and maximum effects of any of the nucleotides tested. Extensive washing with EDTA and the inclusion of 5 mM EDTA in the binding assays also failed to reveal a requirement for Mg2+ to obtain high affinity binding for glucagon. Finally, when we tested whetherwe could repeat inour laboratory the previous findings with @-adrenergicreceptors such as present in S49 cell membranes (22,35), corpora lutea (36), and turkey erythrocytes (22), we had no difficulty in showing that agonist (isoproterenol) displacement curves were “left shifted” on addition of M e . Thus, the effect of M$+ to promote high affinity binding is quite general, i.e. applied to neurotransmitter receptors, but is not universal, i.e. was not observed with rat liver plasma membrane glucagon receptor. Since the H to L transition was shown to occur with GDP in theabsence of M e , our experimentsshow that activation of N. (with respect to C) is not necessary for obtaining a H to L transition at thereceptor level. It is highly unlikely that, under the conditions we used, we were quantitatively activating N,, for this is indeed a reaction that with our membranes is totally dependenton Mg2+ addition (5). It follows that N, must have at least two domains that can change conformation or state independently of each other. We call these domains r and c to describe that one interacts with receptor while the otherreacts with the catalyst Cof the system. We define r+ as the conformation or state of the r domain of N. that confers high affinity binding to thereceptor and r- the conformation or state that exists when receptor exhibits low affinity binding for hormone. It is not totally clear at this point whether r- also implies low affinity of N. for R. In analogy we call c- and c+ the inactive and active forms or states of the c domain of N.. Our experiments showed that in the presence of HR complex, regardless of M e addition, N, is r+c- and thaton addition of GDP, also regardless of presence of M e , N. is r-c-. In this context, 8adrenergic receptors differ from the glucagon receptor in being more stringent in their requirementfor Mg‘+, for only in the combined presence of Mg and absence of guanine nucleotides is it possible to observe an H state of a receptor, i.e. a r+cform of N.. Clearly, our definition of these domains is functional, their structural correlates on the N. protein may be separate or overlap and still need to be defined. Our third finding is that the potency (E&) of GDP in promoting the r+ to r- (and concomitantly the H to L ) transition was about 10-fold higher than that of GTP and approximately 100-fold higher than that of GMP-P(NH)P. Since GDP is more potent than GTP and since for both nucleotides the incubation conditions were such that, atmost, 20% was of a nature other than that added, the 10-fold higher potency of GDP than GTPcannot have been due to conversion of the diphosphate to the triphosphate, regardless of possible presence of a nucleosidediphosphate kinase postulated to be “near” adenylyl cyclase systems by Kimura and collaborators (37, 38). Our finding that GTP analogs are less potent than GTP in causing the H to L transition of the glucagon receptor-N. complex agrees with similar findings by Stadel andLefkowitz (18), with @-adrenergicreceptor-N, system. The requirement for Mg2+ for formation of the H state in the latter system, precludes the testing of the action of

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GTP without conversion to GDP by N, itself. Theimportantandrather unexpected question that emerges from these studies is whether GTP or its analog GMP-P(NH)P affect binding at all. We attempted to obtain independent information on this question by testing for the effect of cholera toxin. Treatment with this toxin is generally considered to be inhibitory to the GTP to GDP transformation that occurs at N. under hormonal influence (15,39). We therefore reasoned that if GTP were ineffective in promoting the H to L transition of the receptor ( i e . the r+ to r- transition at N.) and that if some of the effect of the added GTP were due to “locally” produced GDP, then the potency of GTP in the toxin-treated system should be markedly decreased. This indeed was found. However, although to much lesser degree, the potencies of GDP and GMP-P(NH)P were also decreased. Even though one might have expected that the toxin treatment should not affect the potency of GDP and GMP-P(NH)P, the finding that an effect on the potencies of these “non-hydrolyzable” nucleotides was found, is consistent with the finding of Rodbell and collaborators (40) and Vaughan and collaborators (41) showing that treatment with cholera toxin enhances the rate at which preloaded [3H]GDP can be exchanged for unlabeled guanine nucleotide. Of significance to our question is therefore the finding that toxin treatment resulted in a reduction in the potency of GTP that isbetween 5- and 7-fold more than that of either GDP or GMP-P(NH)P. This is consistent with the idea that theeffect of GTP was decreased by the toxin due to a decreased formation of GDP. Based on the above reasonings and experimental results, we suggest that GDP and not GTP, orone of its analogs, is responsible for causing the r+ to r- transition at N. and the concomitant H to L transition of the receptor. It follows that for N., the c+ conformation of the c domain coexists with the r+ conformation of the r domain, i.e. that after hormone, guanine nucleotide, and M e mediated activation of N., it is in a r+c+ form. It is possible that at thispoint the Bycomplex has dissociated from the rest of the N, protein. However, our experiments do not speak to thateffect. An alternative way of analyzing the results is to assume that N. has two guanine nucleotide binding sites: one responsible for activation of adenylyl cyclase, i.e. for regulation of the c domain, andthe other responsible for causing the receptor to adopt the H or L states, i.e. for regulation of the r domain. In this case, occupancy of the c site by GTP would suffice for causing activation of the c domain, of the r site by GDP would suffice for regulation of the r domain and both sites would be occupied when GTP regulates r the domain of N, or GDP regulates the c domain, as occurs when glucagonmediated stimulation of adenylyl cyclase is elicited (22, 33). Consistent with the view that two classes of guanine nucleotidesites may operate in an adenylyl cyclase system are kinetic data stemming from studies on ADP-ribosylation of N, by cholera toxin (42), on GDP-mediated hormonal stimulation of adenylyl cyclase pre-activated with GMP-P(NH)P (33) and on comparative aspects of dose-response curves of GMP-P(NH)P acting on adenylyl cyclase uersus acting on prelabeled glucagon receptor (43). However, studies with pure N, by Northup et al. (44) as well as by us5, have not revealed the existence of more than one nucleotide binding site/mol of N., even though such preparations ofN, appear to be fully competent and active. They are able to induce the formation of the guanine nucleotide-sensitive H state of @-adrenergic receptors (12,13)and tohydrolyze GTP in response to agonist occupancy of the @-adrenergicreceptor (12, 13). On the basis

’Codina, J. and L. Birnbaumer, unpublished observations.

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Regulation of Glucagon Receptor Binding

of this information, we feel that it isunlikely that our results showing a dissocation between the potencies of GDP, GTP, and GMP-P(NH)P in promoting hormonal activation of Ns and those affecting hormone binding are due to thepresence of two distinct classes of guanine nucleotide binding sites on N.. Rather, we feel that they point atofundamentaldifference in the specificity of the effects these nucleotides have on N, when interacting with the single thus far biochemically uncovered site for guanine nucleotide on N.. In summary, our experiments indicate that on thorough testing, with a suitablemembrane system, we cannot substantiate thewidely held opinion that GTPor its analog promotes the H to L transition of the receptor concurrently with activation ofN, from c- to c+. Rather, it appears that GDP instead of GTP is responsible for the H to L transition and that N, exists in at least three states, each with two independently regulated domains, a r+c- state, which stabilizes receptors in their H state, andis inactive with respect to C, a r+c+state, obtained on addition of both GTP and Mg”, which still stabilizes receptor in its H state, but which is not active in stimulating the catalyst C, and a r-c- state which forms either uia GTP hydrolysis to GDP or on occupancy of the guanine nucleotide binding site by exogenously added GDP, and which is inactive in either stabilizing the receptor in its H state or in stimulating the activity of the catalyst C.M$+, which is required for stabilization of some receptors in their H state, is not required for this event with all receptor systems. REFERENCES 1. Rodbell, M. (1980) Nature 2 8 4 , 17-22 2. Kent, R. S., De Lean, A., and Lefkowitz, R. J. (1980) Mol. Pharmacol. 17, 14-23 3. De Lean, A., Stadel, J. M., and Lefkowitz, R. J. (1980) J. Biol. Chem. 2 5 5 , 7108-7117 4. Howlett, A. C., Sternweis, P. C., Macik, B.A., Van Arsdale, P. M., and Gilman, A. G . (1979) J. Biol. Chem. 254,2287-2295 5. Iyengar, R., and Birnbaumer, L. (1981) J. Biol. Chem. 256, 11636-11641 6. Ivenear. R.. and Birnbaumer., L. (1982) . . Proc. Natl. Acad. Sci. U. ” S.2. 79,’5179-5183 7. Bird, S. J., and Maguire, M. E. (1978) J. Biol. Chem. 253,88268834 8. Williams, L. T., Mullikin, D., and Lefkowitz, R. J. (1978) J. Biol. Chem. 253,2984-2989 9. Limbird, L. E., Gill, D.M., and Lefkowitz, R. J. (1980) Proc. Natl. Acad. Sci. U. S. A. 7 7 , 775-779 10. Iyengar, R. (1981) J. Biol. Chem. 2 5 6 , 11042-11050 11. Limbird, L. E., and Lefkowitz, R. J. (1970) Proc. Natl. Acad. Sci. U. S. A. 75,228-232 12. Brandt, D. R., Asano, T., Pedersen, S. E., and Ross, E. T. (1983) Biochemistry 22,4357-4362 13. Cerione, R. A,, Codina, J., Benovic, J. L.,Lefkowitz, R. J., Birnbaumer, L., and Caron, M. C. (1984) Biochemistry 23, 4519-4525 14. Cassel, D., and Selinger, Z. (1976) Biochim. Biophys. Acta 452, 538-551 15. Cassel, D., and Selinger, Z. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,3307-3311

16. Cassel, D., and Selinger, Z. (1978) Proc. Natl. Acad. Sci. U. S. A. 75,4155-4159 17. Michel, T., and Lefkowitz, R. J. (1982) J. Biol.Chem. 257, 13557-13563 18. Stadel, J. M., and Lefkowitz, R. J. (1981) J. Cyclic Nucl. Res. 7, 363-374 19. Birnbaumer, L., Swartz, T. L., Abramowitz, J., Mintz, P. W., and Iyengar, R. (1980) J. Biol. Chem. 255,3542-3551 20. Iyengar, R., Abramowitz, J., Bordelon-Riser, M., and Birnbaumer, L. (1980) J. Biol. Chem. 255, 3558-3564 21. Rodbell, M., Krans, H.M. J., Pohl, S. L., and Birnbaumer, L. (1971) J. Biol. Chem. 246, 1872-1876 22. Iyengar, R., Abramowitz, J., Bordelon-Riser, M., Blume, A. J., and Birnbaumer, L. (1980) J. Biol. Chem. 2 5 5 , 10312-10321 23. Rojas, F. J., Swartz, T. L., Iyengar, R., Garber, A. J., and Birnbaumer, L. (1983) Endocrinology 1 1 3 , 711-719 24. Bearer, C. F., Knapp, R. D., Kaumann, A. J., Swawrtz, T. L., and Birnbaumer, L. (1980) Mol. Phurmucol. 17,328-338 25. Walseth, T. F., and Johnson, R. A. (1979) Biochim. Biophys. Acta 5 6 2 , 11-31 26. Birnbaumer, L., Torres, H. N., Flawia, M. M., and Fricke, R. F. (1979) Anal. Biochem. 9 3 , 124-133 27. Johnson, G . L., and Bourne, H. R. (1977) Biochem. Biophys. Res. Commun. 78,792-798 28. Salomon, Y., Londos, C., and Rodbell, M. (1974) Anal. Biochem. 58,541-548 29. Bockaert, J., Hunzicker-Dunn, M., and Birnbaumer, L. (1976) J. Biol. Chem. 251,2653-2663 30. Neville, D. M., Jr. (1968) Biochim. Biophys. Acta 154, 540-552 31. Pohl, S. L., Birnbaumer, L., and Rodbell, M. (1971) J. Biol. Chem. 246,1849-1856 32. Salomon, Y., and Rodbell, M. (1975) J. Biol. Chem. 250, 72457250 33. Iyengar, R., and Birnbaumer, L. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,3189-3193 34. Rodbell, M., Lin, M.C., and Salomon, Y. (1974) J. Biol. Chem. 249,59-65 35. Iyengar, R., Bhat, M. K., Riser, M. E., and Birnbaumer, L. (1981) J. Biol. Chem. 256,4810-4815 36. Abramowitz, J., Iyengar, R., and Birnbaumer, L. (1982) Endocrinology 110, 336-346 37. Kimura, N., and Shimada, N. (1983) J. Biol. Chem. 258, 22782283 38. Kimura, N., and Johnson, G . S. (1983) J. Biol.Chem. 2 5 8 , 12609-12617 39. Aktories, K., Schultz, G . , and Jakobs, K. H.(1982) FEBS Lett. 146,65-68 40. Lad, P. M., Nielsen, T. B., Preston, M. S., and Rodbell, M. (1980) J. Biol. Chem. 255,988-995 41. Burns, D.L., Moss, J., and Vaughan, M. (1982) J. Biol. Chem. 257,32-34 42. Gill, D. M., and Meren, R. (1983) J. Biol.Chem. 258, 1190811914 43. Lad, P.M., Welton, A. F., and Rodbell, M. (1977) J. Biol. Chem. 252,5942-5946 44. Northup, J. K., Smigel, M. D., and Gilman, A. G . (1982) J. Biol. Chem. 257, 11416-11423 45. Sternweis, P. C., Northup, J. K., Smigel, M. D., and Gilman, A. G . (1981) J. Biol. Chem. 2 5 6 , 11517-11526 46. Northup, J. K., Sternweis, P. C., and Gilman, A. G . (1983) J. Biol. Chem. 258, 11361-11368 47. Northup, J. K., Smigel, M. D., Sternweis, P. C., and Gilman, A. G . (1983) J. Biol. Chem. 2 5 8 , 11369-11376 48. Scatchard, G . (1949) Ann. N. Y.Acad. Sci. 51,660-672

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Regulation of Glucagon Receptor Binding Supplerenc t o : Regulation of Glucagon Receptor Binding. tack of Effect of Ug and Preferential Role for 0P.I by Yranciaco J. Rojae and Lufr Birnbau-r

EXPERIMXNTAL PROCEDURES Radiochemicals: C1251-Tyr101Monoidoglucagon was s y n t h e s i z e df r o m glucagon a n d c a r r i e r - f r e e lZ51 uaingIodogen. an the o x i d i z i n g a g e n t a n d t h e n p u r i f i e d (HPLCI over C18-uBondapac b y reverse p h a s e h i g h p r e s e u r e l i q u i d c h r o m o t o g r a p h y c o l u m s a p d e s c r i b e dr e c e n t l y( 2 3 1 . Glucagon MIJ a generous g i f t o f W. Bromer, [ E l iL i l l y Company. I n d i a n y 2 5 1 n d . l . L12511Hydroxybenzyliodopindolol was s y n t h e s i z e df r o mc a r r i e r - f r e e I u s i n gc h l o r a m i n e T as t h eo x i d i z i n g by revaree p h a s e HPLC over CIe-uBondapac BLI d e s c r i b e d 1 2 4 1 . a g e n ta n dp v r i fe d was p u r c h a s e df r o mI a o t e xD i a g n o e t i c a .F r i e n d s r o o d ,T e x a e . C a r r i e r - f r e eI 2 $ I ueing a sp t h e s i z e d b y the method Of W a l e e t h a n d J o h n e o n( 2 5 1 C ~ 3 2 P l A T ~ 2 w a New York N Y I . c a r r i e r -P f r. e e su p l i e d by C i n t i c h e mI n c o r p r a t e d( T u x e d o S y n t h e t i c C ~ ~ 3 2 P ? A Twas P purifiedbychromatqraphy 0ver'DEAE-Seph;dex A-25 ( 2 6 1 . G D P p S was i n i t i a l l y a g e n e r o u sg i f tf r o m Dr. F r i t z E c k a t e i n I n a x P l m c kI m t i t u t e .G d t t i n g e n , West G e m n y l a n d was s u b s e q u e n t l yp u r c h a s e df r o m B o e h r i n g a r - M a n n h e i m( E l k h a r t ,I n d . 1 GDP a n d GTP were from Sigma c h e m i c a l Co. 1st. L o u i e ,M i s s o u r i ) ; GMP-PINHIP a n d AMP-PINHIP were f r o mB o e h r i n g e r Mannheim All n u c l e o t i d e s were p u r i f i e d by i o ne x c h a n g ec h r m a t o g r a p h y ( E l k h a r t ,I n d . 1 . b e f o r eb e i n gu s e d1 2 6 1 .c r e a t i n ep h o e p h o k i n a s e .m y o k i n a s ea n dc r e a t i n ep h o s Company a n dp u r i f i e df r o m" G T P - l i k e ' p h a t e Were p u r c h a s e df r o ms i g m ac h e m i c a l c o n t a m i n a n t 8 a* d e s c r i b e d( 2 2 1 . cholera t o x i n was p u r c h a s e df r o ms i g m ac h e m i c a l c ~ . .a c t i v a t e d by t r e a t m e n t with 40 mM DTT 1 2 7 1 .s e p a r a t e df r o m low molecular w e i g h ts u b s t a n c e by c b r o m a t o g r a p h y over Sephader0-25 1191, f r s c t i o n a t a t -70.C u n t i l u s e d . e d i n t o amall s l i q u o t s a n d s t o r e d

Dr.

S p e c i f i cb i n d i n go fI ~ 2 ~ I ] m o n o i o d o g l u c a g o nt ol i v e r membranes(2-4 ug) m e m b r a n ep r o t e i n / 1 0 0 ul a s s a y volume1 was measured as d e s c r i b e d r e c e n t l y ( 2 3 ) usingfiltrationthrough OxoidO f i l t e r s t o s e p a r a t e boundfromunboundlabeled ug membrane p r o b e .S p e c i f i cb i n d i n go f 1251-HYP t o S 4 9 cell membranes(30-40 a polyp r o t e i n / l O O m l a s s a yv o l u m e 1 was measured as d e s c r i b e d ( 2 2 ) u s i n g e t h y l e n e g l y c o lp r e c i p i t a t i o nS t e p t o s e p a r a t e b o u n df r o mu n b o u n dl a b e l e dp r o b e . S p e c i f i cb i n d i n g sa t et h er e s u l to fs u b t r a c t i n gt h ea m o u n to fl a b e l e dp r o b e bound i nt h ep r e s e n c eo f excess u n l a b e l e ds p e c i f i cr e c e p t o rl i g a n d ( 1 . 0 YM glucagon or 1 0 uM p t o p r a n o l o l l f r m t h e m o u n t o f l a b e l e d p r o b e boundunder i d e n t i c a lc o n d i t i o n si nt h ea b s e n c eo fu n l a b e l e dl i g a n d s . Unless s t a t e do t h e r w i s e .i n c u b a t i o n sf o rr e c e p t o rb i n d i n g were a t 32.5' f o r 2 0m i n u t e si nm e d i a c o n t a i n i n g 50 mU h i s - H C 1 , pH 7.6 1.0 ml4 EDTA, 0 . 1 % BSA a n de i t h e r 0.5-0.8 nM [ 1 2 5 1 1 m o n o i ~ d o g l u c a g o nor 0.1 nM 1251-HYP a n d t h e a d d i t i v e s i n d i c a t e d i n t h e l e g e n d st of i g u r e sa n di nt h e text. assays were a8 d e s c r i b e d( 2 2 1i n a f i n a l volume o f 50 ul A d e n y l y lc y c l a s e C O n t a i n i n q 0.5 ml4 AMP-PINHIP a n d2 5 x 106 c m o f lalPha-32P1ATPISDeCifiC

c a m i n a n t s1 2 2 1f o r m e do f i0 mil c r e a t i n e p h o s p h i t e ; 0 . 2 mg/ml c r e a t i n ep h o s p h o mg/m1 myokinase, 25 mn Itis-HC1, pH 7 . 6 , 0.18 BSA, a n dt h e k i n a s ea n d0 . 0 2 text. The reactions were a d d i t i v e sl i s t e di nt h el e g e n d st of i g u r e sa n di nt h e s t o p p e da n dt h e[ 3 2 P l c ~ Pf o r n e d was q u a n t i t a t e da c c o r d i n g t o Sslomon 128) as m o d i f i e d( 2 9 ) .

c.

P r e - t r e a t m e n to fl i v e rm e m b r a n e s (5.0 mg p r o t e i n / m l ) w i t h c h o l e r a t o x i n ug p r o t e i n / m l l was f o r 1 0 m i n u t e s a t 32.5-C i n t h e p r e s e n c e o f 1 0 uM !'%INAD , 1 0 m M MgCl 1 0 mI4 EDTA a n d2 5 mU h i s - H C 1 , pH 7.6. m e t r e a t e d membranes were w a s h e d 3 ' r e s u s p e n d e d a t 5.0 mg p r o t e i n / m l i n 1.0 mU EDTA a n d 10 ml4 h i s - H C 1 , pH 7.6, f r a c t i o n a t e d i n t o a l i g u o t s a n d s t o r e d f o r u p to 2 weka a t -7O'C b e f o r eb e i n gu s e d (19). C o n t r o lm m b r a n e s were t r e a t e di d e n t i c a l l y b u to m i t t i n gc h o l e r at o x i n .

were p r e p a r e d by t h em e t h o do fN e v i l l e R a tl i v e rm e m b r a n e s e db ym h l & (311.

et.

(30) as d e s c r i b -

RESULTS E f f e c t Of MagnesiumandGuanineNucleotides Receptors.

on G l u c a g o nB i n d i n g

t o Glucagon

In v i e wo ft h es t r i k i n ge f f e c to f mgcl on a g o n i s tb i n d i n g t o P - r e c e p t o r s 17.81. 4 i n v e s t i g a t e d t h e p t e n t i a l role O t Uq2+ On b i n d i n g Of t h e p e p t i d e membranes. To h o r m n e[ 1 2 5 1 ] m o n 0 i o d o g l u c a g ~ ~t o glucagon r e c e p t o r so fl i v e r our s u r p r i s e ,r e g a r d l e s so fw h e t he f f e c t so f Mg2+ yere t e s t e d on t h eb i n d i n g o fi n c r e a s i n gc o n c e n t r a t i o n so f1 f S 5 1 1 m o n o i o d q l u c a g o na n da n a l y z e d by t h e m e t h o do fS c a t c h a r d( F i g . 11. or w h e t h e r t h e e f f e c t o f Mg was t e s t e d on t h e glucagon t o d i s p l a c e t h e b i n d i n g o f a f i x e d low c a p a c i t yo fu n l a b e l e dn a t i v e c o n c e n t r a t i o n Of I ~ ~ ~ I l m o n o i o d o g l u c a g o n ( F i g21, . ye f a i l e d t o o b s e r v e an e f glucagon r e c e p t o r f o r it8 l i g a n d s . The e r p e r i f e c t Of Mg on t h e a f f i n i t y o f mentshown o n F i g . 1 was c a r r i e d a t 32.5.C a n di nt h ea b s e n c eo fa d d e dg u a n i n e on P i 2 Was c a r r i e dO u ta t 22-C, t o allow us t o test n u c l e o t i d eT . h a st h o w n f O r a p o t e n t i a l e f f e c t Of Mg2?'beth i n t h e a b s e n c e a n d t h e p r e s e n c e Of g u a n i n e The lower i n c u b a t i o nt e m p e r a t u r e was c h o s e nb e c a u s e we h a dp r e v i nucleotides. o u s l y n o t e d( 2 3 1t h a tn u c l e o t i d ea d d i t i o n reduces a f f i n i t y o f r e c e p t o r s i n a t h i sr e d u c t i o ni na f f i n i t y is t e m p e r a t u r e - d e p e n d e n t manner, and t h a t a t 32.5.C. toolargeto allow f o r a r e l i a b l em e a s u r e m e n to fg u a n i n en u c l e o t i d e - a f f e c t e d 1 1 2 ~ I l m o n o i o d o g l u c a g ~ nb i n d i n g . We t h e r e f o r ep e r f o r m e d several e x p e r i m e n t sa t of 2ZDC, a t e m p e a t u r e a t w h i c h g u a n i n e n u c l e o t i d e a d d i t i o n r e d u c e s b i n d i n g 0.4-0.6 nM 1 ~ 2 5 1 1 m o n o i o d o g l u c a g o nb e t w e e n 6 0 and 758. As s h o r ni nF i g . 2, a l t h o u g h 1 0 0 uM GMP-PINHIP b o t hr e d u c e dt h ea m o u n to f[ 1 2 5 1 1 m o n o i o d o g l u c a g o n by 70-758 a n d i n c r e a s e d t h e c o n c e n t r a t i o n o f u n l a b e l e d glucagon n e c e s s a r y t o was e q u a l l yi n d i s p l a c e1 ~ 2 ~ I l m o n o i o d o g l u c a g o nb y a f a c t o ro f1 2 ,n g 2 +i o n e f f e c t i v ei na l t e r i n gr e c e p t o rb i n d i n gi nt h ep r e s e n c e Of GMP-PINHIP a n di n its absence. T h ee x p e r i m e n to fF i g . 2 was c a r r i e d o u t u s i n g a f i x e dC o n c e n t r a t i o no f W e n e x tt e s t e dw h e t h e r Mq2+ i o nw h i l en o th a v i n g an e f g u a n i n en u c l e o t i d e . glucagon l o r C1~511mo~~iodoglucsgon). had the g u a n i n e n u c l e o t i d e s k n o m t o a f f e c t b i n d i n g . To d o B O w e needed t o C o n t r o lt h el e v e l so fa d d e dg u a n i n en u c l e o t i d e s 3 i l l u s t r a t e s f o r GDP t h a t i n c u b a t i o no fh i g h t h r o u g h o u tt h ei n c u b a t i o n s .F i g . l e v e l s o fl i v e rm e m b r a n e s results i n a marked lose o f a d d e d GDP a f t e r 10 min o f i n c u b a t i o n even when AMP-P(NH1P. which is k n o m t o d e c r e a a e n u c l e o t i d a s e m e d i a t e db r e a k d o w no fg u a n i n en u c l e o t i d e s( 3 2 1 ,h a db e e na d d e d . This loss of GDP t h r o u g h o u t i n c u b a t i o n c o u l d be p a r t i a l l y O v e r C o m e b y s a t u r a t i o n o f t h e d e g r a d i n gp r o c e 8 sb yi n c r e a s i n gt h ec o n c e n t r a t i o : .> fa d d e d GDP when no Mg was R e d u c t i o n i n l e v e l s o f lia d d e d ,b u tC o u l dn o t , when 5 m MgC12 was p r e s e n t . ver membrane r e s u l t e d in p r e s e r v a t i o n of 90-95% o f t h e a d d e d GDP i n t h e absence o f Mg ion and Of 10-75% i nt h ep r e s e n c eo f Mq i o n ,U s i n g low levels o f l i v e r m e m b r a n e s ,a d d i n g0 . 5 ml AMP-P(NHlP a n d t h e n u c l e o s i d e t r i p h o s p h a t e r e g e n e r a t i n g8 y s t m( p u r i f i e d a 8 d e s c r i b e df r o mc o n t a m i n a t i n g" G T P - l i k e "s u b s t a n c e s ) c y c l a e e a a a y s . GTP levels a t t h e e n do f 10 m i n u t ei n c u b a t i o n s u s e di na d e n y l y l were found t o b e 94-96) a n d 89-931 o ft h o s ea d d e d a t t - 0i nt h ea b s e n c ea n d eh-I. upon t e s t i n gf o rp o t e n t i a l p r e s e n c eo f 5 mM MgC12. r e s p e c t i v e l y( n o t e f f e c t s o f Mg i o n on t h e C o n c e n t r a t i o n - e f f e c t curves o fg u a n i n en u c l e o t i d e act i o n OD b i n d i n g Of C'2511monoiodoglucagonunderConditione where n u c l e o t i d e levels were n o t a l t e r e d s i g n i f i c a n t l y d u r i n g t h e t i m e s o f i n c u b a t i o n s , we Obs e r v e d no e f f e c t s o f Mg, r e g a r d l e s a Of w h e t h e r we t e s t e d f o r e f f e c t On t h e or Of GDP o r GDPPS ( n o r a c t i o n Of GTP and GMP-PINHIP ( i l l u s t r a t e d i n F i g . 4 ) shown) f e e t on t h e a f f i n i t y o f r e c e p t o r f o r an e f f e c t on t h e p r a n c y o f a n y o f

.

OD " i o d o S t u d i e s p u b l i s h e d I" 1971 (211 on t h e e f f e c t s o f n u c l e o t i d e s r e v e a l e d u n e q u i v o c a l l y t h a t GDP as w e l l as GTP a f f e c t s glucagon" b i n d i n gh a d this o a r m e t e r . More r e c e n t l v ( 2 2 1 . we showed t h a tb o t h GDP and i t s analog ;:feci a g o n i s tb i n d i n g - t ob e t a - a d r e n e r g i cr e c e p t o r si n a varietyof s y s t e m si n c l u d i n gt u r k e ye r y t h r o c y t e membranes. However, i n none Of t h e s e i t b er u l e d Out t h a t t h e e f f e c t s o f GDP were n o t d u e t o a limited s t u d i e sC o u l d b u ts i g n i f i c a n tt r a n s p h o s p h o r y l a t i o nr e a c t i o nw h e r e b y GTP p r o d u c e d l o c a l l y Was t h ea c t u a le f f e c t o rr e s p o n s i b l ef o rt h eo b s e r v e dc h a n g ei nr e c e p t o rb i n d i n g p r o p e r t i e s .H a v i n ge s t a b l i s h e dc o n d i t i o n sw h e r eb e t t e rt h a n 9 0 % o ft h ea d d e d g u a n i n en u c l e o t i d e i s r e c o v e r e d a s Such a tt h ee n do fi n c u b a t i o n s . we d e t e r m i n curves Of GDP, GTP and GMP-PINHIP O n r e c e p t o rb i n d i n g . e dc o n c e n t r a t i o n - e f f e c t assay i n c l u d e d 0 : .5 m M AMP-P(NH1P. 1 . 0 uM E O T A , 5.0 n!H T h e s eC o n d i t i o n so f MgC12, 1.0 mn CAMP. O . l $ - B S A , 50 mH Tris-HC1, pH. 1 . 6 , and when GTP was s t u d "Experimental i e d ,t h en u c l e o s i d et r i p h o s p h a t er e g e n e r a t i n gs y s t e ml i s t e du n d e r Procedures'.Suchexperiments,showed a n o r d e ro fp o t e n c i e so f GDP(2.S = GDP > GTP > GMP-PINHIP. w i t h GMP-PINHIP and GTP, r e s p e c t l v e l y ,b e i n ga b o u t 100 and 1 0t i m e s less p o t e n t t h a n GDP i n a f f e c t i n g glucagon b i n d i n g .

GDibk

EffectofNucleotides

on Glucagon S t i m u l a t i o n o f A d e n y l y l

Cyclase.

u e i n gi n c u b a t i o nc o n d i t i o n et h a t are i d e n t i c a l t o t h o s e u s e d i n b i n d i n g assayswiththeexceptionthattracemountsof [alpha-32P1ATP we.* added. W e m n i t o r e d t h e e f f e c t s Of GOP, GTP a n d GMP-P(NH)P on l i v e r m e m b r a n ea d e n y l y l cyclaseactivityintheabsenceandinthepresenceof0.4 nn C12511monoiodoglucagon a n d 50 n~ glucagon I t h e l a t t e r t o e l i c i t f u l l s t i m u l a t i o n o f t h e SYBt a l . In a g r e e m e n tw i t hp r e v i o u sr e p o r t s( 2 2 . 3 3 , 3 4 ) GTP and GMP-PINHIP. b u tn o t the absence of g l u c a g o n , a n d a l l t h r e e n u c l e o t i d e s GDP, s t i m u l a t e d a c t i v i t y i n stimulatedactivityinthepresenceof either glucagon or t h em o n o i o d o g l u c a g o n (0.5 n ~ d)e r i v a t i v e .I na g r e e m e n t a180 w i t hp r e v i o u ae t u d i a st h a tu s e da a t u r a t i n gc o n c e n t r a on8 o fg l u c a g o n( 2 0 1 .b o t hs a t u r a t i n gn a t i v eg l u c a g o na n d t o a f f e c t , i n a s i g n i f i c a n t manner. e u b s a t u r a t i n gC 1 % I m n o i o d c q l u c a g o nf a i l e d GTP or GMP-P(NK1P a f f e c t e d the enzyme. F u r t h e r ,t h e t h e EC50 Values Withwhich w l t hw h i c h GDP e n h a n c e dh o r m o n a ls t i m u l a t i o n was e s s e n t i a l l y t h e 8me at low or a t h i g hC o n c e n t r a t i o n so fh o r m o n e .T h u s ,i nt h r e es e p a r a t ee x p e r i m e n t e , EC 0 v a l u e s f o r GMP-P(NHlP r a n g e df r o m0 . 1 2 t o 0 . 3 5 UH, f o r GTP from 0.09 to 0.21 UM a n df o r GDP f r o m0 . 1 5 t o 0 . 2 0 un. These value^ valuea agree w i t h i n a factorof 2 withthoseObtainedearlierat 32.5.C i n s t e a d o f a t 22.C aa waB d o n eh e r e( 2 2 . 3 3 1 . Effects of

cholera T o x i n o n G l U C a q O n B i n d i n q .

NAD+ f o l l o w e d bywashT r e a t m e n to fl i v e r m e m b r a n e sw i t hc h o l e r at o x i na n d i n g , t o e l i m i n a t e excess NAD+. MgC12 a n d c h o l e r a t o x i n , r e s u l t e d i n membranee w i t he s s e n t i a l l yu n a l t e r e db i n d i n go fC 1 2 5 1 1 m o n o i d o g l u c a g o n when compared t o membranes t r e a t e di d e n t i c a l l yb u to m i t t i n gc h o l e r at o x i n .F u r t h e r , as i l l u s tratedinFig. 6, c h o l e r a t o x i n h a d no v i s i b l e e f f e c t on t h e p o s i t i o n o f t h e c o n c e n t r a t i o n - e f f e c t curve w i t h w h i c h u n l a b e l e d g l u c a g o n d i s p l a c e s l a b e l e d glucagon. As b e f o r e , the r e s u l t s were u n a l t e r e d by a d d i t i o n Of MgC12.

We t e s t e dw h e t h e ri n d e e d we h a d. i n t o x i c a t e d .t h em e m b r a n e s by m e a s u r i n g it t o a c t i v i t y t h e i r G T P - e t i m u l a b l e a d e n y l y l cyclase a c t i v i t y andcomparing m a x i m 1s t1i m u l a t e d We found (not shown) t h a t membranes t r e a t by GMP-P(NBIP. ed w t h C h o l e r a t o x i n s h w e d normal responses t o GMP-P(NH)P and MgCl2 notaba lag i n t h e t i n e course o f CAMP a c c u m u l a t i o no ft h e enzyme l yt h ep r e s e n c eo f exposed to GMP-PINHIP a t 5.0 mM Mg2+ a n dr e d u c t i o ni nt h i sl a ga n de n h a n c e m e n t Of s t e a d y S t a t e v e l o c i t y upon i n c r e a s i n g M92+ t o 25 ml4 a n dt h e yr e s p o n d e d In t h e s ee x p e r i m e n t s we also cont o GTP w i t hm a x i m a l CAMP a c c u m u l a t i o nr a t e s . f i r m e d our p r e v i o u s o b s e r v a t i o n t h a t t o x i n t r e a t m e n t d o e s n o t i n t e r f e r e w i t h a t which GMP-P(NHlP Stirnulatheknorneffect Of hormone t o a c c e l e r a t e t h e r a t e Thus, t o t h e e x t e n t t h a t it i e p o s s i b l e to xte8 a d d e n y l y lc y c l a s e ( 3 5 , 2 0 1 . r e s u l t e di ns t i m u l a t i o n cess t h i sP a r a m e t e r ,t h ec h o l e r at o x i nt r e a t m e n th a d o f most, i f n o t a l l , t h ea d e n y l y l cyclase a c t i v a t i n g g u a n i n en u c l e o t i d e - b i n d i n g I t f o l l o w st h e r e f o r et h a tf a i l u r e t o o b s e r v e e f f e c t s on N p 2 g r o t e i nu n i t s . [ I l m o n o i o d o g l u c a g o nb i n d i n g was n o td u e t o i n c o m p l e t e. i n t o x i c a t i o n o. f nembranes.

-"-J

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--

E f f e c t so fC h o l e r aT o x i nT r e a t m e n to fL i v e rM e m b r a n e s R e g u l a t i o no fR e c e p t o rB i n d i n q .

on G u a n i n e N u c l e o t i d e

In c o n t r a s t t o f i n d i n g s i n t h e a b s e n c e o f n u c l e o t i d e . t o x i n t r e a t m e n t a f a manner t h a t Was depenf e c t e dn u c l e o t i d er e g u l a t i o n Of r e c e p t o r b i n d i n g , i n d e n t on t h en u c l e o t i d et e s t e d .T h i s is i l l u s t r a t e di nF i g .7 ,w h i c h summari z e st h er e s u l t so f several e x p e r i m e n t s I. n c u b a t i n gm e m b r a n e sw i t h 0.6-0.8 nM resence [1251monoiodoglucagon. we f o u n dt h a tt o x i nt r e a t m e n tr e s u l t e di nt h e o f 1 0 0 un GMP-PINHIP, i n a 2- t o 2 . 3 - f o l di n c r e a s ei nt h ea m o u n t Of I~2511monoi o d o g l u c a g o nb o u n dw i t h a c o n c o m i t a n t3 - f o l dd e c r e a s ei nt h ec o n c e n t r a t i o no f u n l a b e l e d glucagon r e q u i r e df o r5 0 %i n h i b i t i o no f[ 1 2 5 1 1 n o n o i o d o g l u c a g o nb i n was o n l y a m e r e1 . 8 - f o l di n c r e a s e d i n g . However, t h e e f f e c t o f t o x i n t r e a t m n t a 2 - f o l dd e c r e a s ei nt h ep o s i t i o n Of t h e d i s p l a c e m e n t curve inlabelbundand when t h e a d d e d n u c l e o t i d e was 100 uM GTP a n d was e s s e n t i a l l y a b s e n t when t h e e x p e r i m e n t was c a r r i e d O u t i n t h e p r e s e n c e o f 1 0 0 uM GDP. I D view Of t h e e f f e c t o f t o x i n t r e a t m e n t t o i n c r e a s e b i n d i n g o f s u b s a t u r a t i n g c o n c e n t r a t i o n s Of [ 1 2 5 1 1 m o n o i o d o g l u c a g o ni nt h ep r e s e n c eo f 1 0 0 un GMPP1NH)P b u t n o t i n i t s a b s e n c e , we i n v e s t i g a t e dw h e t h e rt h i si n c r e a s ei nb i n d l n g was a s s o c i a t e dw i t h an i n c r e a s ei n n u m b e ro fs p e c i f i cb i n d i n gs i t e s .F i g . 8 illustratestheresults Of two e x p e r i m e n t sw h e r e M S t u d i e db i n d i n g Of i n c r e a s to c o n t r o la n dt o x i n - t r e a t e d memi n gc o n c e n t r a t i o n so f1 1 2 5 1 ] m o n o i o d o g l u c a g o n b r a n e si nt h ep r e s e n c e Of 1 0 0 U M GMP-PINHIP ( e x p e r i m e n t Of P a n e l A I a n di nt h e 8). A s can be seen, t h ei n a b s e n c eo fa d d e dn u c l e o t i d e( e x p e r i r e n to fP a n e l crease i nb i n d i n go f low c o n c e n t r a t i o n so f1 ~ 2 ~ I l m o n o i o d o g l u c a g o n seen i n t h e p r e s e n c eo f GMP-PINHIP upon t o x i n t r e a t m e n t is n o t d u e t o an i n c r e a s e i n t h e t o an i n c r e a s e i n t h e a f f i n i t y o f numberof a v a i l a b l eb i n d i n gS i t e s ,b u tr a t h e r t h er e c e p t o rf o rt h e h o r m o n e .S i n c et h ea f f i n i t yo fI ' 2 5 I I m o n o i o d o g l u c a g o nf o r t o x i n t r e a t e d membranes vas a b o u t t h e same r e g a t d l e s so fw h e t h e r GMP-P(NH1P had b e e na d d e d ,t h i se x p e r i m e n ts u g g e s t e dt h a tt h ee f f e c to ft o x i nt r e a t m e n tm i g h t b e one o f' u n c o u p l i n g .o ft h en u c l e o t i d ee f f e c t .T h i s ,t o g e t h e rw i t ht h ef a c t t h a tt h ee f f e c to ft o x i nt r e a t m e n t on t h e a c t i o n o f a f i x e dc o n c e n t r a t i o no f v a r y w i t ht h en u c l e o t i d ea d d e d ,l e d us t o t e s t w h e t h e r n u c l e o t i d ea p p e a r e dt o a d e c r e a s ei nt h ep o t e n c i e sw i t hw h i c ht h et h r e e nuct o x i na c t i o nr e s u l t e di n l e o t i d e sa f f e c tb i n d i n g .T h i s was t e s t e di ne x p e r i m e n t s ,s u c h as i l l u s t r a t e d i nF i g . 9 , w h e r er e p r e s e n t a t i v er e s u l t sf r o m t w o s u c he x p e r i m e n t s are shown. I n o n ee x p e r i m n t , ue d e t e r m i n e dt h ec o n c e n t r a t i o n - e f f e c t curves f o r t h e a c t i o n o f GDP and GHP-P(NH1P on 1 1 2 5 1 1 m o n o i o d o g l u c a g ~ "b i n d i n gu s i n g assay c o n d l t i o n s 0.5 mn AMP-P(NH)P a n do m i t t e d a n u c l e o s i d e t r i p h o s p h a t e regenerW h i c hi n c l u d e d a t i n g s y s t e m IRSI 9 0 as t o p r e s e r v e 90-95% o ft h ea d d e d GDP and GMP-P(NH1P as s u c h .T h eo t h e re x p e r i m e n td e t e r m i n e dt h ec o n c e n t r a t i o n - e f f e c tr e l a t i o n s h i p f o rt h ea c t i o n Of GTP and GMP-PINHIP on b i n d i n g u n d e r i d e n t i c a l c o n d i t i o n s b u t i n c l u d i n gt h e RS SO a5 t o p r e s e r v e GTP and GMP-PINHIP l e v e l s . T h ed o u b l e m e a s u r e m e n to ft h ee f f e c t so fv a r y i n gc o n c e n t r a t i o n so f GMP-PINHIP--once i nt h e a b s e n c ea n d once i nt h ep r e s e n c eo fR S - - p r o v i d e d a c o n t r o lt ot e s tf o rp o s s i b l e curves. AS can be e f f e c t s o f RS on t h ep o s i t i o no ft h ec o n c e n t r a t i o n - e f f e c t seen, RS h a s l i t t l e e f f e c t on t h i sp a r a m e t e r . On t h eo t h e rh a n d .t o x i nt r e a t m e n ts h i f t e dt h ec o n c e n t r a t i o n - e f f e c t Curve foT GDP 1 1 - f o l d , t h a t f o r GTP values from two Such 7 7 - f o l da n dt h a tf o r GMP-P(NH)P a b o u t2 0 - f o l d .A v e r a g e GDP, GTP and GMP-PINHlP, e x p e r i m e n t s Yere 1 2 - f o l d ,8 2 - f o l da n d1 8 - f o l df o r respectively.

7834

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Regulation of Glucagon ReceptorBinding

light, the d m " " &e &t into pieces and piwere axulted for d i s t r i l t i m of 32P in a l i q u i d s c i n t i l l a t i m oounter to quantify the f a t e of the added [~-~~P]GOP.

Regulation of Glucagon Receptor Binding Liwer Membranes plus a~'2'1]Monoiodo~luca~on(22°C)

7835