Abstract

The structure of the alpha 1-adrenergic receptor was investigated by comparing polypeptides identified by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis with the size of the intact receptor in cell membranes as determined by target size analysis. The alpha 1-adrenergic receptor from rat liver membranes affinity-labeled with [3H]phenoxybenzamine, a covalent affinity reagent, appeared as a single polypeptide with a molecular mass of 85,000 daltons (Da) on NaDodSO4-polyacrylamide gels. In the absence of protease inhibitors, smaller peptides of 58-62 kDa and 40-45 kDa, specifically labeled with [3H]phenoxybenzamine, were also apparent on NaDodSO4 gels. In order to determine whether the 85-kDa protein represented all or only a portion of the alpha 1-receptor, radiation inactivation (target size analysis) was undertaken. Radiation-induced receptor inactivation was measured by the loss of specific [3H]phenoxybenzamine and [3H]prazosin binding and by the loss of affinity-labeled alpha 1-adrenergic receptors on NaDodSO4 gels. Target size analysis of rat liver alpha 1-receptors indicated that the intact membrane-bound receptor has an average molecular mass of 160,000 Da. These data suggest that the intact alpha-receptor may exist in the membrane as a dimer of two 85,000-Da subunits. The structure of the alpha 1-receptor was further studied by limited proteolysis of the 85-kDa protein isolated from NaDodSO4 gels. Trypsin, chymotrypsin, and papain produce smaller peptides similar to those produced during membrane isolation in the absence of protease inhibition. Limited proteolysis of the membrane-bound receptor produces water-soluble peptides, the largest of which is 45,000 Da. This peptide contains the ligand-binding domain and protrudes from the membrane into the extracellular space.