The proteinase activated receptor-2 (PAR-2) mediates mitogenic responses in human vascular endothelial cells. Molecular characterization and evidence for functional coupling to the thrombin receptor

Proteolytically cleaved receptors, typified by the functional thrombin receptor (TR), represent a novel class of receptors that mediate signaling events by functional coupling to G proteins. Northern blot analysis completed with a human proteinase activated receptor-2 (PAR-2) cDNA as probe demonstrated the similar to 3.5 kb PAR-2 transcript in total cellular RNA from human umbilical vein endothelial cells (HUVEC). Microspectrofluorimetry using Fura2-loaded HUVEC demonstrated a dose-dependent elevation in intracellular calcium transients ([Ca super(2+)] sub(i)) to murine PAR super(39-44) (SLIGRL, putative neoligand after cleavage), with an similar to EC sub(50) of 30 mu M, and evidence for homologous desensitization with complete recovery at 45 min. Xenopus oocytes microinjected with TR cRNA failed to respond to 200 mu M PAR super(39-44), and TR-targeted antisense oligonucleotides specifically abrogated thrombin-induced but not PAR super(39-44)-mediated [Ca super(2+)] sub(i), excluding the possibility that TR/PAR-2 cell-surface coexpression was structurally linked. HUVEC incubated with PAR super(39-44) demonstrated a dose- and time-dependent mitogenic response similar to that seen with thrombin or TR super(42-47) (TR-activating peptide, SFLLRN). Preactivation of HUVEC with either PAR super(39-44) or thrombin resulted in heterologous desensitization to the corresponding agonist, an effect that was mediated primarily by TR internalization as evaluated by immunofluorescence and quantitative ELISA. These results ascribe a previously unrecognized function to the PAR-2 receptor, imply that a natural enzyme agonist may circulate in plasma, and suggest the presence of an additional regulatory mechanism controlling receptor activation events in vascular endothelial cells.