Localization of the fourth membrane spanning domain as a ligand binding site in the human platelet a2-adrenergic receptor

Hiroaki Matsui, Robert J. Lefkowitz, Marc G. Caron, John W. Regan

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Abstract

The human platelet α2-adrenergic receptor is an integral membrane protein which binds epinephrine. The gene for this receptor has been cloned, and the primary structure is thus known [Kobilka et al. (1987) Science 238, 650–656]. A model of its secondary structure predicts that the receptor has seven transmembrane spanning domains. By covalent labeling and peptide mapping, we have identified a region of the receptor that is directly involved with ligand binding. Partially purified preparations of the receptor were covalently radiolabeled with either of two specific photoaffinity ligands: [3H]SKF 102229 (an antagonist) or p-azido[3H]clonidine (an agonist). The radiolabeled receptors were then digested with specific endopeptidases, and peptides containing the covalently bound radioligands were identified. Lysylendopeptidase treatment of [3H]SKF 102229 labeled receptor yielded one peptide of Mr 2400 as the product of a complete digest. Endopeptidase Arg-C gave a labeled peptide of Mr 4000, which was further digested to the Mr 2400 peptide by additional treatment with lysylendopeptidase. Using p-azido[3H]clonidine-labeled receptor, a similar Mr 2400 peptide was obtained by lysylendopeptidase cleavage. This Mr 2400 peptide corresponds to the fourth transmembrane spanning domain of the receptor. These data suggest that this region forms part of the ligand binding domain of the human platelet α2-adrenergic receptor.

Original languageEnglish (US)
Pages (from-to)4125-4130
Number of pages6
JournalBiochemistry
Volume28
Issue number9
DOIs
StatePublished - May 1989
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry

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