Abstract
Fusion proteins were generated between the human 5-hydroxytryptamine (5-HT)1A receptor and both wild-type (Cys351) and pertussis toxin-resistant (Gly351 and Ile351) forms of Gi1. These were expressed stably. Pertussis toxin treatment substantially reduced basal high-affinity GTPase activity in clones expressing the 5-HT1A receptor wild-type Gi1α construct but not in clones expressing 5-HT1A receptor (Gly351)Gi1α or (Ile351)Gi1α. Spiperone functioned as an inverse agonist in membranes expressing the 5-HT1A receptor wild-type Gi1α fusion protein and in those expressing 5-HT1A receptor (Ile351)Gi1α but not the 5-HT1A receptor (Gly351)Gi1α fusion protein. The effect of spiperone at the 5-HT1A receptor wild-type Gi1α construct but not the 5-HT1A receptor (Ile351)Gi1α construct was blocked by pertussis toxin treatment. By contrast, agonists functioned with equal effectiveness at the three fusion proteins and were unaffected by pertussis toxin treatment of the (Ile351)Gi1α- and (Gly351)Gi1α-containing constructs. 5-HT resulted in strong inhibition of forskolin-amplified adenylyl cyclase in intact cells expressing the isolated 5-HT1A receptor. In fusion protein-expressing cells, 5-HT-mediated inhibition of adenylyl cyclase was also observed. Pertussis toxin treatment obliterated 5-HT-mediated inhibition in cells expressing the isolated receptor and the 5-HT1A receptor wild-type Gi1α fusion protein but not in those expressing the 5-HT1A receptor (Ile351) or (Gly351)Gi1α fusion proteins. These studies demonstrate that alteration of a single amino acid in Gi1α located at a key contact site between the G protein and a G protein-coupled receptor can regulate agonist-independent constitutive activity of the G protein-coupled receptor and that fusion proteins can directly regulate adenylyl cyclase.
Footnotes
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Send reprint requests to: Dr. Graeme Milligan, Davidson Bldg., University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, United Kingdom. E-mail:g.milligan{at}bio.gla.ac.uk
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This work was supported by the Medical Research Council and the European Union Biomed II program: Inverse agonism: Implications for drug design.
- Abbreviations:
- 5-HT
- 5-hydroxytryptamine
- GPCR
- G protein-coupled receptor
- MPPF
- 4(2′-methoxy)-phenyl-1-[2′-(N-2"-pyridinyl)-p-fluorobenzamido]ethyl-piperazine
- OH-DPAT
- hydroxy-2-(di-n-propylamino)tetralin
- PCR
- polymerase chain reaction
- HEK
- human embryonic kidney
- PAGE
- polyacrylamide gel electrophoresis
- Received April 1, 1999.
- Accepted June 24, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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