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(Received for publication, October 9, 1996, and in revised form, November 18, 1996)
,
,
,¶
From the Mutations in several domains can lead to
agonist-independent, constitutive activation of G protein-coupled
receptors. However, the nature of the structural and molecular changes
that constitutively turn on a G protein-coupled receptor remains
unknown. Here we show evidence that a constitutively activated mutant
of the 
Howard Hughes Medical Institute and the
¶ Division of Cardiovascular Medicine, Stanford University Medical
School, Stanford, California 94305 and the § Department of
Physiology and Biophysics, Mount Sinai School of Medicine,
New York, New York 10029
2 adrenergic receptor (CAM) is characterized by
structural instability and an exaggerated conformational response to
ligand binding. The structural instability of CAM could be demonstrated
by a 4-fold increase in the rate of denaturation of purified receptor
at 37 °C as compared with the wild type receptor. Spectroscopic
analysis of purified CAM labeled with the conformationally sensitive
and cysteine-reactive fluorophore,
N,N
dimethyl-N-(iodoacetyl)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine, further indicated that both agonist and antagonist elicit more profound structural changes in CAM than in the wild type protein. We
propose that the mutation that confers constitutive activity to the
2 adrenergic receptor removes some stabilizing
conformational constraints, allowing CAM to more readily undergo
transitions between the inactive and the active states and making the
receptor more susceptible to denaturation.
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