RT Journal Article
SR Electronic
T1 The Critical Role of Transmembrane Prolines in Human Prostacyclin Receptor Activation
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 1202
OP 1210
DO 10.1124/mol.61.5.1202
VO 61
IS 5
A1 Jeremiah Stitham
A1 Kathleen A. Martin
A1 John Hwa
YR 2002
UL http://molpharm.aspetjournals.org/content/61/5/1202.abstract
AB The human prostacyclin receptor (hIP), a G protein-coupled receptor (GPCR), plays important roles in vascular smooth muscle relaxation as well as the prevention of platelet aggregation. It has been postulated that GPCR transmembrane (TM) prolines serve as molecular hinges or swivels and are necessary for proper binding and activation. By individually (as well as collectively) mutating these hIP prolines to alanine, the ability to form key structural and functional configurations was removed. Significant effects on both binding and activation were observed. Two highly conserved prolines across GPCRs, Pro-154, and Pro-254 (TMVI), showed the greatest effect on decreasing both binding and activation when changed to alanine. Along the extracellular boundary of the highly conserved transmembrane III domain, a proline-to-alanine mutation at position 89 (P89A) revealed normal binding affinity in comparison with the 1D4-epitope–tagged hIP (hIP1D4) wild-type control (K i, iloprost = 3 ± 2 versus 7 ± 3 nM, respectively). In contrast, activation was markedly affected, with an EC50 of 12.0 ± 2.5 nM compared with that of 1.2 ± 0.3 nM (10-fold difference) for the hIP1D4. Movement within TMIII has been shown to be necessary for effective GPCR activation. Both the extracellular location (above the putative binding pocket) along with an exclusive effect upon activation suggest that this movement is facilitated by the presence of Pro-89 and independent from the actions of ligand binding. This finding strongly supports a model in which proline residues serve as molecular hinges or swivels, essential for coupling receptor binding to activation.