Abstract
Immunoprecipitation of a fusion protein between the α1b-adrenoceptor and Gα11 following a [35S]GTPγS [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]GTPγS/mol of fusion protein. This was to the G protein linked to the receptor rather than endogenous Gαq/Gα11 as a fusion protein containing the α1b-adrenoceptor and a form of Gα11 (G208A) unable to exchange guanine nucleotides effectively, bound [35S]GTPγS very poorly. Fusion proteins between A293E, D142A, and 3CAM mutants of the α1b-adrenoceptor and Gα11bound substantially greater levels of [35S]GTPγS 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]GTPγS and acted as a partial inverse agonist for each of the constitutively active mutants. [35S]GTPγS binding to Gα11 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 α subunit of a Gq family G protein and defines a strategy potentially suitable for any receptor that couples to these G proteins.
Footnotes
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Financial support for this work was provided by the Medical Research Council.
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Current address: Scottish Biomedical, Todd Campus, West of Scotland Science Park, Glasgow G20 OXA, Scotland, UK.
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DOI: 10.1124/jpet.102.035501
- Abbreviations:
- GPCR
- G protein-coupled receptor
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- CAM
- constitutively active mutant
- PCR
- polymerase chain reaction
- PAGE
- polyacrylamide gel electrophoresis
- WB4101
- 2-(2,6-dimethoxyphenoxyethyl)aminomethyl 1,4-benzodioxane
- HV723
- α-ethyl 3,4,5-trimethoxy-α-(3-[2-(2-methoxyphenoxy)ethyl]}propyl)benzeneacetonitrile fumarate
- Received February 28, 2002.
- Accepted April 9, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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