Abstract
The ligand binding pocket of biogenic amine G protein-coupled receptors is embedded in the membrane-spanning regions of these receptors, whereas the extracellular domains of the peptidergic receptors play a key role in the structure and function of this class of receptors. To examine the role of the extracellular sequences in prostaglandin receptor-ligand interaction, chimeras were constructed with the two Gs-coupled E-prostanoid (EP) receptors, replacing each of the extracellular sequences of the human EP2receptor with the corresponding human EP4 receptor residues. Replacement of the third extracellular loop (ECIII) yielded a receptor that binds [3H]prostaglandin E2(PGE2; K d = 6.3 nM) with similar affinity as the EP2 wild-type receptor (K d = 12.9 nM). Similarly, replacement of the nonconserved carboxyl-terminal portion of ECII resulted in a receptor that maintains [3H]PGE2 binding (K d = 8.8 nM). In contrast, replacement of the amino terminus, ECI, the entire ECII region, or the residues within the highly conserved motif of the amino-terminal half of ECII yielded chimeras that displayed neither detectable [3H]PGE2 binding nor receptor-evoked cAMP generation. Immunoprecipitation demonstrated that each chimera is expressed at levels near that of wild-type receptors; however, enzyme-linked immunosorbent assay revealed that inactive chimeras have reduced cell surface expression. Similarly, chimeras that exchange the multiple extracellular loop sequences N/ECI, ECII/ECIII, or all four sequences lacked detectable binding and signal transduction, and although expressed, were not detected on the cell surface. These data suggest that the extracellular sequences of the EP2receptor are critical determinants of receptor structure and/or function, unlike other G protein-coupled receptors that bind small molecules.
Footnotes
- Received October 26, 1998.
- Accepted May 14, 1999.
-
Send reprint requests to: Dr. Richard M. Breyer, Division of Nephrology, S3223 MCN, Vanderbilt University, Nashville TN 37232-2372. E-mail:rich.breyer{at}mcmail.vanderbilt.edu
-
Support for this project was provided in part by National Institutes of Health Grants DK46205 (R.M.B.), GM15431 (R.M.B.), DK37097 (M.D.B.), Cancer Center Support Grant CA68485, and a United States Pharmacopoeia Predoctoral Fellowship Award (B.A.S.).
- 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.
|