[125I-Tyr10]Monoiodoglucagon ([125I]MIG) was cross-linked to liver membrane glucagon receptors with hydroxysuccinimidyl-p-azidobenzoate, and the products were analyzed by sodium dodecyl sulfate-gel electrophoresis. Autoradiograms of the gel obtained after a 24-h exposure showed one major band at M(r) = 63,000 that was sensitive to GTP and excess unlabeled glucagon. Exposure for 7 days showed labeling of an additional M(r) = 33,000 species that was also sensitive to excess unlabeled glucagon. The M(r) = 33,000 peptide can be obtained by subtilisin, trypsin, elastase, or Staphylococcus aureus V8 protease treatment of the [125I]MIG-occupied receptor in the membrane or in Lubrol-PX solution. In contrast, limited proteolysis of membranes containing vacant receptors results in labeling of a M(r) = 24,000 peptide. The M(r) = 24,000 peptide specifically binds [125I]MIG in a GTP-sensitive manner. The M(r) = 33,000 peptide also retains GTP sensitivity since it releases bound [125I]MIG upon addition of GTP. Elastase treatment of the electroeluted M(r) = 33,000 peptide yields the M(r) = 24,000 and 15,000 fragments. The M(r) = 15,000 peptide is the smallest fragment of the receptor as yet identified. Treatment of the M(r) = 63,000 receptor with [125I]MIG cross-linked to it with endo-β-N-acetylglucosaminidase F results in four distinct fragments with M(r) values of 61,000, 56,000, 51,000, and 45,000; prolonged treatment resulted in the accumulation of the last two. Neither the M(r) = 33,000 nor the M(r) = 24,000 fragment appeared to be substrates for endo-β-N-acetylglucosaminidase F. These data indicate that glucagon receptor is a glycoprotein of ~60,000 daltons which contains at least four N-linked glycans accounting for 18,000 daltons of its mass. Both its glucagon binding function and its capacity to interact with the stimulatory regulator of adenylyl cyclase are containted within a fragment of only ~21,000 daltons that does not contain any N-linked glycans. Hormone occupancy of the receptor results in a conformational change so as to expose a region that is susceptible to proteolysis by proteases of varying specificities to yield a peptide of ~30,000 daltons that also does not contain N-linked glycans.
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1984|