Coupling of the alpha 1-adrenergic receptor to a guanine nucleotide-binding regulatory protein by a discrete domain distinct from its ligand recognition site

• Published in: Biochimica et biophysica acta Vol. 968; no. 1; p. 119
• Main Authors: Graham, R M, Sena, L M, Longabaugh, J P, Sawutz, D G, Schwarz, K R, Homcy, C J
• Format: Journal Article
• Language: English
• Published: Netherlands 18-01-1988
• Subjects: Animals; Binding Sites; Cell Membrane - metabolism; Epinephrine - pharmacology; Female; GTP-Binding Proteins - metabolism; Guanylyl Imidodiphosphate - pharmacology; Kinetics; Ligands; Liver - metabolism; Macromolecular Substances; Prazosin - metabolism; Protein Binding; Rats; Rats, Inbred Strains; Receptors, Adrenergic, alpha - metabolism
• DOI: 10.1016/0167-4889(88)90051-1
• ISSN: 0006-3002

At rat hepatic membrane alpha 1-adrenergic receptors, the nonhydrolyzable GTP analogue p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding. This p[NH]ppG effect is consistent with the involvement of a guanine nucleotide-binding regulatory protein (G-protein) in alpha 1-adrenergic receptor signalling. Although readily apparent in membranes prepared to avoid retention of endogenous nucleotides and activation of Ca2+-sensitive proteinases (+pi), this p[NH]ppG effect is not observed in membranes prepared without proteinase inhibitors (-pi), or in -pi membranes treated with Ca2+ (-pi, +Ca2+). In these various membrane preparations, different Mr forms of the receptor are also identified by photoaffinity labeling with [125I]CP65526, an aryl azide analog of the alpha 1-selective antagonist, prazosin, followed by SDS-polyacrylamide gel electrophoresis and autoradiography. Whereas a predominant Mr = 80,000 subunit is identified in +pi membranes, in -pi membranes a proteolytic Mr = 59,000 fragment is also observed. In -pi, +Ca2+ membranes, only this latter peptide is detected. To evaluate the ability of each of these forms of the receptor to couple with a G-protein, the effect of p[NH]ppG on the agonist-inhibition of [125I]CP65526 labelling was determined by laser densitometry scanning and computer analysis. At the Mr = 80,000 subunit, p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding, even in -pi membranes. By contrast, agonist-binding at the Mr = 59,000 subunit is of low-affinity and was not affected by p[NH]ppG. These data indicate that the cleaved Mr = 59,000 fragment, while retaining hormone binding activity is unable to undergo G-protein coupling. Thus, the alpha 1-adrenergic receptor appears to contain a discrete domain necessary for G-protein coupling that is distinct from its ligand recognition site.