Ravi Iyengar

Regulation of Hormone Receptors and Adenylyl Cyclases by Guanine Nucleotide Binding N Proteins

Publisher Summary Receptors that affect cyclic adenosine monophosphate (cAMP) are sub-classified into two subtypes: Rs receptors, which increase cAMP levels by stimulating the enzyme adenylyl cyclase, and Ri receptors, which decrease cAMP levels by inhibiting the cAMP-forming enzyme. This chapter discusses the transduction mechanism to which Rs- and Ri-type receptors couple to modulate adenylyl cyclase activity. At the center of this transduction mechanism are two oligomeric coupling proteins called N or G proteins. These proteins have properties to bind and hydrolyze guanosine triphosphate and regulate hormone affinity for receptors and the catalytic activity of the cAMP-forming enzyme. This complex receptor-coupling protein-adenylyl cyclase system is approached by first reviewing structural and functional aspects that regulate cAMP formation. The chapter also discusses the basic structure and regulation of adenylyl cyclase by nucleotides and magnesium. It also discusses action of hormones on the nucleotide-regulated system. It analyzes the known regulation of hormone-receptor interaction by the coupling proteins. The analysis of affinity regulation of receptors leads to conclusions that point toward the existence of at least two conformational states of receptors interacting with at least three conformational states or forms of the coupling proteins.

Guanyl nucleotide regulation of hormonally-responsive adenylyl cyclases.

A large number of hormones and neurotransmitters activate adenylyl cyclase [ATP, pyrophosphate lyase (cyclizing; EC 4.6.1.1.)] catalyzing the formation of cAMP and PPi from ATP in the presence of Mg2+. The cAMP formed is in turn responsible for eliciting the physiological responses of these hormones and neurotransmitters. In addition to hormones and neurotransmitters, fluoride ion, cholera toxin and guanyl nucleotides (GTP and GTP analogs such as GTP gamma S and GMP-P(NH)P) also stimulate adenylyl cyclase activity (Perkins, 1974; Birnbaumer, 1977; Gill, 1977). It has become evident that hormonally-responsive adenylyl cyclase is a multi-component system consisting of at least 3 physically distinct units. The first is the hormone receptor containing a specific site for a given hormone. The second is the catalytic moiety (C component) of adenylyl cyclase bearing the site responsible for catalysis of the cyclizing reaction. The third is the guanyl nucleotide regulatory subunit (G component) which binds guanyl nucleotide. Recently, a GTPase activity has been found to be associated with the G component of adenylyl cyclase (Cassel and Selinger, 1976; Cassel et al., 1977a, b; Lambert et al., 1979). In this review we will present information on the regulation of hormonally-responsive adenylyl cyclases. This is not intended to be a comprehensive review of the literature. Rather, it represents our views on the current status of the regulation of cAMP formation.

Covalent labeling of the hepatic glucagon receptor

Publisher Summary The procedure for covalent labeling the hepatic glucagon receptor utilizes the light-sensitive heterobifunctional cross-linker hydroxysuccinimidyl- p -azidobenzoate (HSAB) to link the bound [ 125 I-Tyr l0 ]monoiodoglucagon ([ 125 I]MIG) to the receptor protein. The procedure was described to covalently attach 125 I -labeled glucagon to its liver membrane receptor. The method involves first the binding of the labeled hormone to its receptor and the removal of the excess unbound label. This is then followed by incubation with the crosslinker, in the dark and then under ultraviolet illumination to covalently couple the bound [ 125 I] MIG. The chapter also explains that HSAB contains an amino reactive group and an aryl azide, which, upon light activation, is converted to an aryl nitrene that reacts in a chemically unspecific manner. The chapter discusses various methods for covalent labeling of the hepatic glucagon receptor such as [ 125 I] MIG binding to liver membranes, cross-linking of bound [ 125 I] MIG to liver membranes, and SDS-gel electrophoretic analysis of covalently labeled membranes.

Wheat germ lectin-sepharose affinity adsorption assay for the soluble glucagon receptor.

Publisher Summary This chapter explains that the hepatic glucagon-sensitive adenylyl cyclase has been widely used as a model system to study the signal transduction process in adenylyl cyclase systems. However, purification of the receptor has not been possible up to now. This is due to lack of availability of a reliable assay for the solubilized receptor. The chapter also explains that the methods commonly used for other soluble receptors, such as gel filtration and polyethylene glycol precipitation, have not proven useful for assaying the glucagon receptor due to unacceptably high backgrounds. The chapter attempts to develop a simple assay that would be based on a unique property of the receptor and explains that in the past few years, it has been found that many hormone receptors are glycoproteins and the hormone–receptor complex could be specifically adsorbed onto the lectin-Sepharose, while the free hormone would remain in solution. The solid phase containing the hormone–receptor complex could be separated from the free hormone by low-speed centrifugation and removal of the supernatant. The hormone–receptor complex formed could then be estimated by counting the lectin-Sepharose in a gamma counter.

Hormonal Regulation of Adenylyl Cyclase Activity

Peptide and protein hormones such as glucagon and gonadotropins and neurotransmitters such as chatecholamines exert their action on target cells by binding to their respective receptors (R). These interactions lead to stimulation of cAMP formation by the adenylyl cyclase systems in these cells. In what follows we shall review and present key experimental evidences on functional aspects of cAMP forma- tion by adenylyl cyclases as seen both in the absence and presence of hormonal influence. We shall present current knowledge on the molecular composition and structure of hormone sensitive adenylyl cyclases. Taking structural as well as functional aspects into account we shall discuss current thoughts on how both hormonal stimulation and the ensuing desensitization to hormonal stimulation come about. Finally we shall present some speculations as to other forms of regulation, especially attenuation of cAMP formation and raise some of the most pertinent questions in signal transduction research.

Solubilization of an angiotensin II binding site from rat liver

The high affinity binding sites for angiotensin II were solubilized from rat liver membranes by treatment with CHAPS. The binding protein was also partially purified by angiotensin III inhibitor-coupled Affi-gel affinity chromatography. Binding to the intact membranes as well as to the solubilized preparation was specific and saturable. According to the Scatchard plot, the membrane preparations exhibited a single class of high affinity binding sites with aKd of 0.71 nM. The solubilized preparation also showed the presence of a single class of binding sites with less affinity (Kd of 14 nM). Meanwhile the competition studies using angiotensin II analogues represented two separate binding sites for angiotensin II and single binding site for antagonist. These latter findings were correlated to the results provided by Garrisons research group. More works are needed to clarify this discrepancy.

CHARACTERIZATION OF THE HEPATIC GLUCAGON RECEPTOR

The hepatic glucagon receptor was covalently labeled with [125I-Tyr10]-monoiodoglucagon by use of the heterobifunctional crosslinker hydroxysuccinimidyl-p-azidobenzoate and analyzed by SDS-gel electrophoresis. The autoradiogram of the gel showed one band at Mr = 63,000 that was sensitive to excess unlabeled glucagon and GTP. The labeled receptor was solubilized with Lubrol-PX and the hydrodynamic characteristics of the receptor were determined. The molecular parameters of the solubilized receptor are S20,w = 4.3 +/- 0.1, Stokes radius = 6.3 +/- 0.1 nm, frictional coefficient f/f degrees = 1.8 and a calculated Mr = 119,000. Incubation of liver membranes at 32 degrees for 15 min prior to the addition of [125I-Tyr10] permitted us to identify the high molecular weight form (Mr approximately equal to 113,000) by direct SDS-gel electrophoretic analysis. Limited elastase treatment of the hormone-occupied receptor results in the appearance of a Mr = 33,000 fragment, that retains guanine nucleotide sensitivity. Elastase treatment of vacant receptors results in a Mr = 24,000 fragment that binds hormone in a GTP-sensitive manner. The Mr = 24,000 fragment is contained within the Mr = 33,000 fragment. The Mr = 63,000 receptor upon treatment with endo-beta-N-acetylglucosamine F for 4 h yields four fragments of apparent Mr = 61,000, 56,000, 51,000, and 45,000; 24 h treatment results in the accumulation of the last two fragments. Neither Mr = 33,000 and 24,000 fragment appear to be substrates for endo-beta-N-acetylglucosaminidase F. These data allow us to conclude that the hepatic glucagon receptor in the membrane is a dimer of approximately 60,000 dalton hormone binding subunit which is a glycoprotein containing at least four N-linked glycans accounting for 18,000 daltons of its mass. Both the hormone binding function and the capacity for the interaction with the stimulatory regulator of adenylyl cyclase are contained within a fragment of only approximately 21,000 daltons that does not contain any N-linked glycans.