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J. Biol. Chem., Vol. 277, Issue 23, 21080-21085, June 7, 2002
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From the Department of Cellular and Molecular Physiology, Yale
University School of Medicine, New Haven, Connecticut 06520-8026
To investigate the subcellular organization of
receptor-G protein signaling pathways, a robust dominant negative
A Highly Effective Dominant Negative
s Construct
Containing Mutations That Affect Distinct Functions Inhibits
Multiple Gs-coupled Receptor Signaling Pathways*
s mutant containing substitutions that alter
distinct functions was produced and tested for its effects on
Gs-coupled receptor activity in HEK-293 cells. Mutations in
the 3
5 loop region, which increase receptor affinity,
decrease receptor-mediated activation, and impair activation of
adenylyl cyclase, were combined with G226A, which increases affinity
for 
, and A366S, which decreases affinity for GDP. This triple
s mutant can inhibit signaling to Gs from the luteinizing hormone receptor by 97% and from the calcitonin receptor by 100%. In addition, this s mutant blocks all
signaling from the calcitonin receptor to Gq. These results
lead to two conclusions about receptor-G protein signaling. First,
individual receptors have access to multiple types of G proteins in
HEK-293 cell membranes. Second, different G protein subunits can
compete with each other for binding to the same receptor. This dominant negative s construct will be useful for determining
interrelationships among distinct receptor-G protein interactions in a
wide variety of cells and tissues.
*
This work was supported by National Institutes of Health
Grant GM50369 and American Heart Association Established Investigator Grant 9740043N.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 203-785-3202;
Fax: 203-785-4951; E-mail: catherine.berlot@yale.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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