Coupling of the alpha 1-adrenergic receptor to a guanine nucleotide-binding regulatory protein by a discrete domain distinct from its ligand recognition site.
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.
Graham
R M
RM
Cardiac Unit, Massachusetts General Hospital, Boston 02114.
Sena
L M
LM
Longabaugh
J P
JP
Sawutz
D G
DG
Schwarz
K R
KR
Homcy
C J
CJ
D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D004837:Epinephrine / Q000494:pharmacology; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000378:metabolism*; D046911:Macromolecular Substances; D011224:Prazosin / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*
D008024:Ligands; D046911:Macromolecular Substances; D011942:Receptors, Adrenergic, alpha; D006165:Guanylyl Imidodiphosphate; D019204:GTP-Binding Proteins; D011224:Prazosin; D004837:Epinephrine