Glycosylation of human proteinase-activated receptor-2 (hPAR2): role in cell surface expression and signalling

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Abstract
We have analysed the role of N-linked glycosylation in regulating human proteinase-activated receptor-2 (hPAR2) expression and function. Epitope-tagged wild-type hPAR2 (wt-hPAR2) or hPAR2 that lacked glycosylation sequons (following site-directed mutagenesis) in either the N-terminus [hPAR2N30A (Asn30→Ala)], extracellular loop 2 [ECL2; hPAR2N222Q (Asn222→Gln) or hPAR2N222A (Asn222→Ala)] or both (hPAR2N30A,N222A or hPAR2N30A,N222Q) were expressed in the Chinese-hamster ovary (CHO) fibroblast cell line, Pro5. Western blot analysis of wt-hPAR2 showed mature wt-hPAR2 to have a molecular mass of 55—100kDa, and 33—48kDa following N-glycosidase F deglycosylation. FACS analysis and immunocytochemistry of the wt-hPAR2 and PAR2 mutant cell lines revealed that removal of both glycosylation sequons decreases (50% of wt-hPAR2) cell surface expression. Western blot analysis indicated that both N-linked sites are glycosylated. In functional studies, hPAR2N30A displayed a selective and significant increase in sensitivity towards tryptase. Interestingly, hPAR2N222A displayed a loss in sensitivity towards all PAR2 agonists tested. However, further analysis revealed receptor sensitivity to alanine mutations in this domain, as the more conservative substitution hPAR2N222Q displayed no change in response to PAR2 agonists. hPAR2N30A,N222Q displayed increased sensitivity towards tryptase, but a loss in sensitivity towards trypsin and the synthetic peptide SLIGRL-NH2, although this loss in sensitivity towards trypsin and SLIGRL-NH2 was secondary to changes in cell-surface expression. Finally, expression of sialic-acid-deficient wt-hPAR2 in the CHO Lec2 glycosylation-deficient mutant cell line, showed a 40kDa loss in molecular mass, in addition to a marked and selective increase in sensitivity towards tryptase. We conclude that hPAR2 N-linked glycosylation and sialylation regulates receptor expression and/or signalling.