Abstract
The effects of Mg2+ and guanine nucleotides on glucagon binding to its receptor were studied using [125I-Tyr10]monoiodoglucagon. Contrary to findings with beta-adrenergic receptors, high affinity binding of the stimulatory hormone was not dependent on Mg2+ and low affinity binding could be obtained on nucleotide addition regardless of presence of Mg2+. GDP, guanyl-5'-yl thiophosphate (GDP beta S), GTP, and guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) were all able to induce low affinity hormone binding. Since the Ns component of adenylyl cyclase, with which the receptor interacts, is inactive in stimulating the catalytic component C of adenylyl cyclase in the absence of Mg2+, both before and after GDP addition, it is suggested that Ns has at least two domains that change conformation independently of each other: a r domain, that interacts with the receptor and confers to it high affinity binding, and a c domain, that interacts with the catalyst C and stimulates it. It is suggested further that Ns is r+c- when stabilizing the receptor in its conformation with high affinity for hormone, and r-c- when under the influence of GDP which results in the receptor adopting the conformation that exhibits low affinity for the hormone. Comparison of potencies of the four nucleotides to induce low affinity binding showed that GDP and GDP beta S were equipotent and 10 times more potent than GTP and 100 times more potent than GMP-P(NH)P. Under the conditions used it was impossible to substantiate that the effects of GTP or GMP-P(NH)P were not due to formation of GDP from GTP or presence of GDP-like material in GMP-P(NH)P. It is suggested that, contrary to widely held opinions, GDP and GDP-like compounds, and not GTP or its analogs, are responsible for the lowering of the affinity of adenylyl cyclase stimulating receptors for their hormones or agonists. Furthermore, the experiments suggest that the c+ conformation of the c domain of Ns co-exists with the r+ and not the r- conformation of its r domain.
Highlights
The effectsof Mg2+and guanine nucleotides on glu- Studies carried out largely with the P-adrenergic receptor, but cagon binding to its receptor were studiedusing [1261- to some extent with other receptors, have shown that the
Since . component of adenylyl cyclase, with which the receptor interacts, is inactive in stimulating the catalyticcomponent C of adenylyl cyclase in the absence of M e +, both before and after GDP addition, it is suggested that N. has at least two domains that change conformation independdescribed and so have the activation ofN. byM$+ and guanine nucleotides andthe stimulation of the activation process by agonist-receptor complexes
1.)Thesestudies led to the current recognition that both receptor and N, exist in at least two forms or states: receptors in HandL states, exhibiting high and low affinity for agonists [2, 3], and N. in inactive (NScoa)nd active (Ns(a)) states,of which the active state is able to enhance the catalytic activity ently 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 [4,5,6].* Correlation of intrinsic c domain, that interacts with the catalyst C and stim- activity of a large number of catecholamine analogs having ulates it
Summary
The abbreviations used are: N., stiinulatory N; AMP-P(NH)P, The active form of N. may merely be a different conformation of adenyl-5”yl imidodiphosphate; BSA, bovine serum albumin; GMP- the N protein or, as suggested by studies with non-hydrolyzable PNH2,guanyl-5’-yl imidophosphate; GDPDS, guanyl-5’-yl thiophos- analogs, represent the 01 subunit complexes with the guanine nucleophate; GMP-P(NH)P, guanyl-5”yl imidodiphosphate, C, catalytic tide unseparated from the 07 portion of the protein [44,45,46,47]. Regulation of GlucagBoindRiencgeptor bines with this complex and leads to formation of HR(L)plus
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