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 ~ 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 ([Ca²⁺](i)) to murine PAR^39-44 (SLIGRL, putative neoligand after cleavage), with an ~ EC₅₀ of 30 μM, and evidence for homologous desensitization with complete recovery at 45 min. Xenopus oocytes microinjected with TR cRNA failed to respond to 200 μM PAR^39-44, and TR-targeted antisense oligonucleotides specifically abrogated thrombin- induced but not PAR^39-44-mediated [Ca²⁺](i), excluding the possibility that TR/PAR-2 cell-surface coexpression was structurally linked. HUVEC incubated with PAR^39-44 demonstrated a dose- and time-dependent mitogenic response similar to that seen with thrombin or TR^42-47 (TR- activating peptide, SFLLRN). Preactivation of HUVEC with either PAR^{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.