Role of extracellular disulfide-bonded cysteines in the ligand binding function of the. beta. sub 2 -adrenergic receptor

Journal Article · Tue Mar 06 04:00:00 EST 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00461a018· OSTI ID:7192964

Dohlman, H G; Caron, M G; DeBlasi, A; Frielle, T; Lefkowitz, R J ^[1]

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1. Duke Univ. Medical Center, Durham, NC (USA)

Evidence is presented for a role of disulfide bridging in forming the ligand binding site of the {beta}{sub 2}-adrenergic receptor ({beta}AR). The presence of disulfide bonds at the ligand binding site is indicated by competitive inhibition by dithiothreitol (DTT) in radioligand binding assays, by specific protection by {beta}-adrenergic ligands of these effects, and by the requirement of disulfide reduction for limit proteolysis of affinity ligand labeled receptor. The kinetics of binding inhibition by DTT suggest at least two pairs of disulfide-bonded cysteines essential for normal binding. Through site-directed mutagenesis, the authors indeed were able to identify four cysteines which are critical for normal binding affinities and for the proper expression of functional {beta}AR at the cell surface. Unexpectedly, the four cysteines required for normal ligand binding are not those located within the hydrophobic transmembrane domains of the receptor (where ligand binding is presumed to occur) but lie in the extracellular hydrophilic loops connecting these transmembrane segments. These findings indicate that in addition to the well-documented involvement of the membrane-spanning domains of the receptor in ligand binding, there is an important and previously unsuspected role of the hydrophilic extracellular domains in forming the ligand binding site.

• OSTI ID: 7192964
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:9; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

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62 RADIOLOGY AND NUCLEAR MEDICINE
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AROMATICS
AZAARENES
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BETA DECAY RADIOISOTOPES
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CHEMICAL BONDS
CYSTEINE
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ISOTOPE APPLICATIONS
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LABELLED COMPOUNDS
LIGANDS
MEMBRANE PROTEINS
MOLECULAR STRUCTURE
MUTANTS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PROTEINS
PYRROLES
RADIOISOTOPES
RADIORECEPTOR ASSAY
RECEPTORS
THIOLS
TRACER TECHNIQUES