Clusters of Transmembrane Residues Are Critical for Human Prostacyclin Receptor Activation

Abstract

Relaxation of vascular smooth muscle and prevention of blood coagulation are mediated by ligand-induced activation of the human prostacyclin (hIP) receptor, a seven-transmembrane-domain G-protein-coupled receptor (GPCR). In this study, we elucidate the molecular requirements for receptor activation within the region of the ligand-binding pocket, identifying transmembrane residues affecting potency. Eleven of 30 mutated residues in the region of the ligand-binding domain exhibited defective activation (decreased potency). These critical residues localized to four distinct clusters (analysis via a rhodopsin-based human prostacyclin receptor homology model). Residues Y75(2.65) (TMII), F95(3.28) (TMIII), and R279(7.40) (TMVII) comprised the immediate binding-pocket cluster and were shown to be essential for proper receptor activation, compared to equivalent expression levels of the wild-type hIP (WT EC(50) = 1.2 +/- 0.1 nM; Y75(2.65)A EC(50) = 347.3 +/- 62.8 nM, p < 0.001; F95(3.28)A EC(50) = 8.0 +/- 0.6 nM, p < 0.001; R279(7.40)A EC(50) = 130 +/- 63.0 nM, p < 0.001). Residues S20(1.39) (TMI), F24(1.43) (TMI), and F72(2.62) (TMII) were localized to a cluster involving P17(1.36), a critical residue thought to facilitate transmembrane movement during changes in activation conformation. A third cluster formed around amino acid D60(2.50) (TMII), containing the highly conserved (100% of prostanoid receptors) D288(7.49)/P289(7.50) motif located in TMVII. Last, a large hydrophobic cluster composed of aromatic residues F146(4.52) (TMIV), F150(4.56) (TMIV), F184(5.40) (TMV), and Y188(5.44) (TMV) was observed away from the ligand-binding pocket, but still necessary for hIP activation. These results assist in delineating the potential molecular requirements for agonist-induced signaling through the transmembrane domain. Such observations may be generally applicable, as many of these clusters are highly conserved among the prostanoid receptors as well as other class A GPCRs.

The First Extracellular Loop of the Saccharomyces cerevisiae G Protein-coupled Receptor Ste2p Undergoes a Conformational Change upon Ligand Binding

“…Previous data with the signaling-compromised 102, 105, 108, 111, and 114 cysteine mutants indicate that alanine substitution is tolerated and results in at least partially active receptors at all but position 111, indicating the Tyr 111 residue is essential for protein function (37). Because the phenotype resulting from mutations at Tyr 266 and Asn 205 are similar to that for Y111C, we postulate that these three residues are part of a hydrophobic pocket that has been described to be involved in ligand activation in other GPCRs (52,53). Because many of the well characterized dominant-negative mutations in Ste2p have been mapped to the junctions between the extracellular loops and the TMs (27), our data provide some insight into this observation.…”

Prostacyclin Analogs Inhibit Fibroblast Contraction of Collagen Gels through the cAMP-PKA Pathway

“…Prostacyclin is known to act, at least in part, through the cAMP-PKA pathway that is activated by prostacyclin binding to the G s -coupled prostacyclin receptor followed by subsequent activation of adenylyl cyclase (11). To investigate whether prostacyclin analogs increase PKA activity in human lung fibroblasts, confluent cells were incubated with the three prostacyclin analogs at the concentration of 1 ϫ 10 Ϫ6 M for 15, 30, and 60 min.…”

The prostacyclin receptor induces human vascular smooth muscle cell differentiation via the protein kinase A pathway

“…PGI 2 mediates its cellular activity via the tissue-specific, cell surface G protein-coupled receptor, the prostacyclin receptor IP (International Union of Pharmacology Receptor classification) (2). IP, expressed on both VSMC and platelets, predominantly couples to the heterotrimeric G protein G s , leading to activation of adenylyl cyclase (AC), formation of the second messenger cAMP, and activation of the cAMP-dependent protein kinase, PKA (13,30). Because prostacyclin is a vasodilator as well as a potent inhibitor of platelet activation and of VSMC proliferation, it is considered an important endogenous factor that protects blood vessels from cardiovascular disease.…”