Rat protease-activated receptor–1 (rPAR1) expression and characterization in Sf9 cells
Thrombin’s serine protease activity allows for it to have a role in both the coagulation cascade as well as through a GTP- binding protein coupled receptor (GPCR) known as the protease-activated receptor-1 (PAR1). Thrombin binds to PAR1 at the N-terminal, cleaving between Arg41 and Ser42, and unmasking a new N-terminal which acts as a tethered ligand. Thrombin and a five amino acid peptide composed of the sequence of residues Ser42 to Arg46, known as PAR 1-AP, has been shown to mediate a number of signalling mechanisms in mammalian cells, including a calcium signalling pathway.    In the present study, the Sf9-baculovirus system allowed us to express the rat PAR1 (rPAR1-Sf9) on the cell surface and study its intracellular signalling.  The calcium (Ca2+) signal was studied using the fluorescent probe Fura-2, and several Ca2+ channel inhibitors and calcium signal modulators were used to study the signal induced by PAR1-AP.    In the presence of extracellular calcium [Ca2+]e, thrombin and PAR1-AP produced a Ca2+ signal which consisted of an initial spike in [Ca2+]i followed by a relatively maintained plateau and a slow return towards baseline.  In the absence of Ca2+ in the extracellular space, the initial Ca2+ increase is followed by a quick return to baseline [Ca2+]i.  Ca2+ channel inhibitors, lanthanum, nifedipine and D-600, inhibited the entry of Ca2+ from the extracellular space and abolished the plateau phase of the response to PAR1-AP. Inhibition of the Ca2+-ATPase with thapsigargin abolished the response to PAR1-AP after having depleted the Ca2+ stores involved in the initial spike in [Ca2+]i. TMB-8, expected to inhibit the release of Ca2+ from internal stores, inconsistently inhibited the [Ca2+]i  response to PAR1-AP. The response elicited by PAR1-AP was not affected by D-609, an inhibitor of phospholipase Cβ. Inhibition of protein kinase C (PKC) with bisindolylmaleimide induced oscillations in the [Ca2+]i levels in the presence of extracellular Ca2+ while it significantly blunted the response in the absence of extracellular Ca2+. PDBu activation of PKC truncated the [Ca2+]i surge in Ca2+-rich conditions while abolishing it altogether in the absence of extracellular Ca2+.  H-89 inhibition of protein kinase A (PKA) prolonged the plateau duration in Ca2+-rich medium while inhibiting the response to PAR1-AP in a Ca2+-deficient environment.   Taken together, our results suggest that PKC and possibly PKA play a critical role in the mobilisation of Ca2+ in rPAR1-Sf9 by PAR1-AP.
baculovirus Fura-2 thrombin protease-activated receptor calcium channel calcium signalling Sf9 cell