Title : Functional consequences of alteration of N-linked glycosylation
sites on the
neurokinin 1 receptor
Abstract :
- The neurokinin 1 receptor ( NK1R ), a G protein-coupled receptor involved in diverse functions including pain and inflammation, has two putative N-linked glycosylation sites, Asn-14 and Asn-18
- We studied the role of N-linked glycosylation in the functioning of the NK1R by constructing three receptor mutants: two single mutants ( Asn --> Gln-14 and Asn --> Gln-18 ) and a double mutant, lacking both glycosylation sites
- Using a lentiviral transfection system, the mutants were stably transfected into NCM 460 cells, a nontransformed human colonic epithelial cell line
- We observed that the magnitude of glycosylation as estimated by changes in gel migration depends on the number of glycosylation sites available, with the wild-type receptor containing the greatest amount of glycosylation
- All mutant receptors were able to bind to substance P and neurokinin A ligand with similar affinities; however, the double mutant, nonglycosylated NK1R showed only half the B( max ) of the wild-type NK1R
- In terms of receptor function, the ablation of both N-linked glycosylation sites did not have a profound effect on the receptors ' abilities to activate the MAP kinase families ( p42 / p44 , JNK , and p38 ), but did affect SP-induced IL-8 secretion
- All mutants were able to internalize, but the kinetics of internalization of the double mutant receptor was more rapid, when compared with wild-type NK1R
- Therefore, glycosylation of NK1R may stabilize the receptor in the plasma membrane
- These results contribute to the ongoing elucidation of the role of glycosylation in G protein-coupled receptors and the study of the neurokinin receptors in particular