Site-directed mutagenesis of the cytoplasmic domains of the human β2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling

B. F. O'Dowd, M. Hnatowich, John W Regan, W. M. Leader, M. G. Caron, R. J. Lefkowitz

Research output: Contribution to journalArticle

295 Citations (Scopus)

Abstract

Numerous plasma membrane-bound receptors are coupled to various effectors via a family of guanine nucleotide regulatory proteins (G proteins). Amino acid sequences of these receptors, deduced from cDNA and genomic clones, indicate the presence of seven transmembrane-spanning domains. Alignment of the available amino acid sequences of these G protein-linked receptors reveals striking homologies in regions predicted to lie near the cytoplasmic surface of the cell membrane. As these areas are likely those which interact with G proteins, we reasoned that systematic introduction of non-native sequence into these highly conserved regions of the human β2-adrenergic receptor would allow resolution of loci participating directly in receptor-G protein coupling. Based on this strategy, we constructed 19 mutant receptor species comprising substitutions and deletions of native sequence in the putative cytoplasmic domains of human β2-adrenergic receptors. By monitoring ligand binding characteristics and receptor-mediated stimulation of adenylyl cyclase, we have determined that the C-terminal portion of the third cytoplasmic loop and the N-terminal segment of the cytoplasmic tail appear to be critical for productive receptor-coupling to G proteins. In addition, we have implicated two other areas of the receptor that possibly play supportive roles in maintaining proper orientation of the G protein binding site. These comprise the second cytoplasmic loop and a conserved cysteine residue in the cytoplasmic tail.

Original languageEnglish (US)
Pages (from-to)15985-15992
Number of pages8
JournalJournal of Biological Chemistry
Volume263
Issue number31
StatePublished - 1988
Externally publishedYes

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Mutagenesis
Site-Directed Mutagenesis
GTP-Binding Proteins
Adrenergic Receptors
Cell membranes
Tail
Amino Acid Sequence
Amino Acid Receptors
Cell Membrane
Amino Acids
Sequence Deletion
Adenylyl Cyclases
Protein Binding
Cysteine
Substitution reactions
Complementary DNA
Clone Cells
Binding Sites
Ligands
Monitoring

ASJC Scopus subject areas

  • Biochemistry

Cite this

Site-directed mutagenesis of the cytoplasmic domains of the human β2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling. / O'Dowd, B. F.; Hnatowich, M.; Regan, John W; Leader, W. M.; Caron, M. G.; Lefkowitz, R. J.

In: Journal of Biological Chemistry, Vol. 263, No. 31, 1988, p. 15985-15992.

Research output: Contribution to journalArticle

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