JPET

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Carrillo, J. J.
Right arrow Articles by Milligan, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Carrillo, J. J.
Right arrow Articles by Milligan, G.

Vol. 302, Issue 3, 1080-1088, September 2002

Measurement of Agonist-Dependent and -Independent Signal Initiation of alpha 1b-Adrenoceptor Mutants by Direct Analysis of Guanine Nucleotide Exchange on the G Protein Galpha 11

Juan J. Carrillo, Patricia A. Stevens and Graeme Milligan

Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom

Immunoprecipitation of a fusion protein between the alpha 1b-adrenoceptor and Galpha 11 following a [35S]GTPgamma S [guanosine-5'-O-(3-thio)triphosphate] binding assay resulted in incorporation of low levels of nucleotide. The agonist phenylephrine increased incorporation some 30-fold. Agonist-induced binding represented 1.0 mol of [35S]GTPgamma S/mol of fusion protein. This was to the G protein linked to the receptor rather than endogenous Galpha q/Galpha 11 as a fusion protein containing the alpha 1b-adrenoceptor and a form of Galpha 11 (G208A) unable to exchange guanine nucleotides effectively, bound [35S]GTPgamma S very poorly. Fusion proteins between A293E, D142A, and 3CAM mutants of the alpha 1b-adrenoceptor and Galpha 11 bound substantially greater levels of [35S]GTPgamma S in the absence of agonist than the fusion incorporating the wild-type receptor. Constitutive binding of the nucleotide induced by these mutants was only 20% of the level achieved by phenylephrine. These mutant receptors thus do not provide an accurate mimic of the agonist-occupied state. Phentolamine reduced the binding of [35S]GTPgamma S and acted as a partial inverse agonist for each of the constitutively active mutants. [35S]GTPgamma S binding to Galpha 11 was elevated by phenylephrine in both wild-type and constitutively active mutant forms of the fusion proteins, but agonist potency and binding affinity were 50 times higher for the fusions containing the mutated receptors. These studies provide the first direct demonstration of the capacity of constitutively active mutants of a receptor to stimulate guanine nucleotide exchange on the alpha  subunit of a Gq family G protein and defines a strategy potentially suitable for any receptor that couples to these G proteins.

0022-3565/02/3023-1080$07.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics


This article has been cited by other articles:


Home page
 Mol. Pharmacol.Home page
L. A. Stoddart, A. J. Brown, and G. Milligan
Uncovering the Pharmacology of the G Protein-Coupled Receptor GPR40: High Apparent Constitutive Activity in Guanosine 5'-O-(3-[35S]thio)triphosphate Binding Studies Reflects Binding of an Endogenous Agonist
Mol. Pharmacol., April 1, 2007; 71(4): 994 - 1005.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
G. Pascal and G. Milligan
Functional Complementation and the Analysis of Opioid Receptor Homodimerization
Mol. Pharmacol., September 1, 2005; 68(3): 905 - 915.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
D. Ramsay, I. C. Carr, J. Pediani, J. F. Lopez-Gimenez, R. Thurlow, M. Fidock, and G. Milligan
High-Affinity Interactions between Human {alpha}1A-Adrenoceptor C-Terminal Splice Variants Produce Homo- and Heterodimers but Do Not Generate the {alpha}1L-Adrenoceptor
Mol. Pharmacol., August 1, 2004; 66(2): 228 - 239.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
A. Heydorn, R. J. Ward, R. Jorgensen, M. M. Rosenkilde, T. M. Frimurer, G. Milligan, and E. Kostenis
Identification of a Novel Site within G Protein {alpha} Subunits Important for Specificity of Receptor-G Protein Interaction
Mol. Pharmacol., August 1, 2004; 66(2): 250 - 259.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. J. Carrillo, J. Pediani, and G. Milligan
Dimers of Class A G Protein-coupled Receptors Function via Agonist-mediated Trans-activation of Associated G Proteins
J. Biol. Chem., October 24, 2003; 278(43): 42578 - 42587.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
F. Bertaso, R. J. Ward, P. Viard, G. Milligan, and A. C. Dolphin
Mechanism of Action of Gq to Inhibit Gbeta gamma Modulation of CaV2.2 Calcium Channels: Probed by the Use of Receptor-Galpha Tandems
Mol. Pharmacol., April 1, 2003; 63(4): 832 - 843.
[Abstract] [Full Text] [PDF]


Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
All ASPET Journals Molecular Pharmacology Pharmacological Reviews
Molecular Interventions Drug Metabolism and Disposition

Copyright © 2002 by the American Society for Pharmacology and Experimental Therapeutics.