Abstract
The mechanism by which receptors activate G proteins is unclear because a connection between the receptor and the nucleotide binding site has not been established. To investigate this mechanism, we evaluated the roles in receptor interaction of three potential receptor contact sites in αs: the α2/β4, α3/β5, and α4/β6 loops. Substitutions of αi2 homologs for αsresidues in the α2/β4 loop and alanine substitutions of residues in the α4/β6 loop do not affect activation by the β2-adrenergic receptor. However, replacement of five αs residues in the α3/β5 loop region with the homologous αi2 residues decreases receptor-mediated activation of αs and increases the affinity of Gs for this receptor. The substitutions do not alter guanine nucleotide binding or hydrolysis, or activation by aluminum fluoride, indicating that the effects on receptor interaction are not due to a destabilization of the guanine-nucleotide bound state. In a model of the receptor-G protein complex, the α3/β5 loop maps near the second and third intracellular loops of the receptor. The effects of the α3/β5 substitutions suggest that the wild-type residues may be receptor contact sites that are optimized to ensure the reversibility of receptor-G protein interactions. Furthermore, the α3/β5 region corresponds to an exchange factor contact site in both EF-Tu and Ras, suggesting that the mechanisms by which seven-transmembrane receptors and exchange factors catalyze nucleotide exchange may share common elements.
Footnotes
-
Send reprint requests to: Dr. Catherine H. Berlot, Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8026. E-mail:catherine.berlot{at}yale.edu
-
This work was supported by National Institutes of Health Grant GM50369 (C.H.B.) C.H.B. is an Established Investigator of the American Heart Association.
-
↵1 Residue numbering throughout is according to the long splice variant of αs.
-
↵2 The αs residue 354 is not shown because it represents an insertion of sequence relative to that of the αt/αi1 chimera in the model.
- Abbreviations:
- GTPγS
- guanosine 5′-O-(3-thiotriphosphate)
- ICYP
- iodocyanopindolol
- DTT
- dithiothreitol
- Received November 9, 1999.
- Accepted February 10, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|