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Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling.

Publication ,  Journal Article
O'Dowd, BF; Hnatowich, M; Regan, JW; Leader, WM; Caron, MG; Lefkowitz, RJ
Published in: J Biol Chem
November 5, 1988

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 beta 2-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 beta 2-adrenergic receptor. 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.

Duke Scholars

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

November 5, 1988

Volume

263

Issue

31

Start / End Page

15985 / 15992

Location

United States

Related Subject Headings

  • Xenopus laevis
  • Receptors, Adrenergic, beta
  • Protein Conformation
  • Protein Biosynthesis
  • Oocytes
  • Mutation
  • Molecular Sequence Data
  • Humans
  • GTP-Binding Proteins
  • Female
 

Citation

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O’Dowd, B. F., Hnatowich, M., Regan, J. W., Leader, W. M., Caron, M. G., & Lefkowitz, R. J. (1988). Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling. J Biol Chem, 263(31), 15985–15992.
O’Dowd, B. F., M. Hnatowich, J. W. Regan, W. M. Leader, M. G. Caron, and R. J. Lefkowitz. “Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling.J Biol Chem 263, no. 31 (November 5, 1988): 15985–92.
O’Dowd BF, Hnatowich M, Regan JW, Leader WM, Caron MG, Lefkowitz RJ. Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling. J Biol Chem. 1988 Nov 5;263(31):15985–92.
O’Dowd BF, Hnatowich M, Regan JW, Leader WM, Caron MG, Lefkowitz RJ. Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling. J Biol Chem. 1988 Nov 5;263(31):15985–15992.

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

November 5, 1988

Volume

263

Issue

31

Start / End Page

15985 / 15992

Location

United States

Related Subject Headings

  • Xenopus laevis
  • Receptors, Adrenergic, beta
  • Protein Conformation
  • Protein Biosynthesis
  • Oocytes
  • Mutation
  • Molecular Sequence Data
  • Humans
  • GTP-Binding Proteins
  • Female