Requirement of atypical N-glycosylation motif for the surface expression and the intracellular signaling

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We currently found that GPR109A, which is known as a nicotinic acid (niacin) receptor, is modified withN-glycan, even through lacking the sequon motif in the extracellular regions. Here, we demonstrate that Asn17-X-Cys^<19>, termed non-sequon motif, within the N-terminus of this receptor contributes to the N-glycosylation of GPR109A. This modification is indispensable for GPR109A because non-glycosylated GPR109A mutants, GPR109A/N17A and GPR109A/C19A, showed impaired cell surface expression, although GPR109A/C19S and GPR109A/C19T mutants, in which receiveN-glycosylation at Asn^<17>, exhibited similar expression to wild-type receptor. We further suggest that the oligosaccharyltransferase catalytic subunit STT3B, but not STT3A isoform, is implicated in theN-glycan conjugation on the non-sequon motif of GPR109A. Intriguingly, defect in the di-cysteine in the non-sequon motif, Cys^<18>-Cys^<19>, resulted in the impairment of the Gi-mediated signaling via GPR109A, e.g., nicotinic acid-elicited reduction of cAMP synthesis and elevation of intracellular [Ca^<2+>]. Since this deficiency was not compensated by the replacement of Cys19 to Ser or Thr, the impaired function of the di-cysteine defective GPR109A is not due to the lack ofN-glycan.