Beta-adrenergic receptors display intramolecular disulfide bridges in situ: analysis by immunoblotting and functional reconstitution.
The molecular nature of mammalian beta-adrenergic receptors in situ was probed using immunoblotting and functional reconstitution techniques. Membrane proteins of cells replete with beta-adrenergic receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were transferred to nitrocellulose and then probed with anti-receptor antibodies. When cell membranes were first treated with agents that cleave disulfides of proteins, immunoblots of these membranes revealed intense immunoreactive bands with electrophoretic mobility similar to that of protein standards of Mr 65,000-67,000, comigrating with purified, reduced, and alkylated beta-adrenergic receptors. However, when cell membranes were prepared under anaerobic conditions, solubilized in the presence of agents that alkylate thiols, and denatured in the absence of added thiols, immunoblotting revealed receptor with Mr 55,000, rather than 65,000. This faster electrophoretic mobility is associated with the presence of intramolecular disulfides in the purified receptor and demonstrates that beta-adrenergic receptors possess intramolecular disulfide bridges in situ. Purified receptors that demonstrate this faster mobility (Mr 55,000 under nonreducing conditions) were co-reconstituted into phospholipid vesicles with the stimulatory GTP-binding protein GS and their ability to catalyze the binding of [35S]guanosine-5'-O-(3-thio)triphosphate to GS was measured. Agonist (isoproterenol) as well as thiol increased the receptor-promoted activation of GS. Taken together, these data demonstrate that native beta-adrenergic receptors possess one or more intramolecular disulfide bridges in situ, reduction of which causes functional activation of the receptor.
Moxham
C P
CP
Department of Pharmacological Sciences, State University of New York, Stony Brook 11794-8651.
Ross
E M
EM
George
S T
ST
Malbon
C C
CC
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure; D004220:Disulfides; D019204:GTP-Binding Proteins / Q000502:physiology; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011943:Receptors, Adrenergic, beta / Q000502:physiology*; D013329:Structure-Activity Relationship
D004220:Disulfides; D046911:Macromolecular Substances; D011943:Receptors, Adrenergic, beta; D019204:GTP-Binding Proteins