Title : Glycosylation of human
proteinase-activated receptor-2 (
hPAR2 ): role in cell surface expression and
signalling
Abstract :
- We have analysed the role of N-linked glycosylation in regulating human proteinase-activated receptor-2 ( hPAR(2) ) expression and function
- Epitope-tagged wild-type hPAR(2) (wt- hPAR(2) ) or hPAR(2) that lacked glycosylation sequons (following site-directed mutagenesis) in either the N-terminus [hPAR(2 )N30A ( Asn(30)--> Ala )], extracellular loop 2 [ ECL2; hPAR(2 )N222Q ( Asn(222)--> Gln ) or hPAR(2)N222A ( Asn(222)--> Ala )] or both ( hPAR(2)N30A ,N222A or hPAR(2)N30A ,N222Q) were expressed in the Chinese-hamster ovary (CHO) fibroblast cell line, Pro5
- Western blot analysis of wt- hPAR(2) showed mature wt- hPAR(2) to have a molecular mass of 55-100 kDa, and 33-48 kDa following N -glycosidase F deglycosylation
- FACS analysis and immunocytochemistry of the wt- hPAR(2) and PAR(2) mutant cell lines revealed that removal of both glycosylation sequons decreases (50% of wt- hPAR(2) ) cell surface expression
- Western blot analysis indicated that both N-linked sites are glycosylated
- In functional studies, hPAR(2)N30A displayed a selective and significant increase in sensitivity towards tryptase
- Interestingly, hPAR(2 )N222A displayed a loss in sensitivity towards all PAR(2) agonists tested
- However, further analysis revealed receptor sensitivity to alanine mutations in this domain , as the more conservative substitution hPAR(2)N222Q displayed no change in response to PAR(2) agonists
- hPAR(2)N30A ,N222Q displayed increased sensitivity towards tryptase, but a loss in sensitivity towards trypsin and the synthetic peptide SLIGRL-NH(2), although this loss in sensitivity towards trypsin and SLIGRL-NH(2) was secondary to changes in cell-surface expression
- Finally , expression of sialic-acid-deficient wt- hPAR(2) in the CHO Lec2 glycosylation-deficient mutant cell line, showed a 40 kDa loss in molecular mass, in addition to a marked and selective increase in sensitivity towards tryptase
- We conclude that hPAR(2) N-linked glycosylation and sialylation regulates receptor expression and/or signalling