Functional consequences of alteration of N-linked glycosylation sites on the neurokinin 1 receptor - PubMed (original) (raw)

Comparative Study

. 2007 Jun 19;104(25):10691-6.

doi: 10.1073/pnas.0703394104. Epub 2007 Jun 11.

Affiliations

Comparative Study

Functional consequences of alteration of N-linked glycosylation sites on the neurokinin 1 receptor

Morris F Tansky et al. Proc Natl Acad Sci U S A. 2007.

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Extent of glycosylation and expression of wild-type and mutant receptors. (A) NK1R immunoblot of transfected NCM 460 cell lysates shows the nonglycosylated mutant receptors migrate at a lower relative mass than the wild-type NK1R, reflecting a loss of carbohydrate moieties. (B) Real-time PCR comparison of lentiviral mutants in NCM 460 cells, demonstrating equal NK1R message for the NCM 460 cells transfected with the various glycosylation mutants (α = 0.05, P > 0.05). NCM 460 NK1R mRNA levels were normalized to TATA-box binding protein mRNA. The values are expressed as a percentage of the wild-type NK1R mRNA abundance.

Fig. 2.

Fig. 2.

Competition binding and 125I-BH-SP saturation analyses of wild-type and glycosylation receptor mutants in NCM 460 cells. These analyses demonstrate that there are no effects of glycosylation on the _K_i of SP in the presence of 125I-BH-SP (A; competition binding analysis), but that there is a 48% decrease in the _B_max of the nonglycosylated mutant when compared with the other mutants and wild-type (B; saturation analysis).

Fig. 3.

Fig. 3.

NK1R immunostaining of NCM 460 cells transfected with wild-type and mutant NK1 receptors, after stimulation by SP for the indicated durations. Untreated cells containing wild-type or mutant receptors show NK1R immunoreactivity on the cell surface, whereas SP stimulation triggers NK1R internalization, visualized as punctate staining (closed arrows). After longer SP stimulation, the mutant receptors are still retained within the cell, in contrast to the wild-type receptors that have returned to the cell surface (open arrows) by 45 min. Representative images of each cell line are shown. (Scale bar: 20 μm.)

Fig. 4.

Fig. 4.

Internalization kinetics of NK-1R mutants. Cells preincubated with 125I-BH-SP for 1 h at 4°C were warmed to 37°C for the indicated times. Cells were acid-washed to remove surface-bound ligand and treated with alkaline wash to determine the amount of internalized ligand by a gamma counter. The double glycosylation receptor mutant displays a significant 24% increase in the relative amount of internalized receptor when compared with the wild-type receptor (α = 0.01, P < 0.01).

Fig. 5.

Fig. 5.

Comparison of SP-induced phosphorylation of MAPK pathways p38, p42/p44, and JNK in cell lines transfected with NK1R mutants. Immunoblot shows the duration of SP-mediated activation of p38, p42/p44, and JNK in all cell lines. p38 and p42/p44 are phosphorylated within 5 min of SP incubation, whereas JNK phosphorylation occurs within 30 min. Wild-type and mutant receptors exhibit similar phosphorylation patterns. Total p42/p44 (shown), total p38, and total JNK were also assayed to assure loading equivalency. Representative immunoblots from independent experiments are shown.

Fig. 6.

Fig. 6.

Immunoblots demonstrating SP-specific effect of MAPK phosphorylation, using NK1R antagonist CJ12,255. Pretreatment of cells with the NK1R antagonist ablates p42/p44 and JNK phosphorylation induced by SP (purple: SP alone; orange: antagonist plus SP) at the indicated times, suggesting that SP acts solely through NK1R to activate the MAPK family.

Fig. 7.

Fig. 7.

Transfected NCM 460 cells were stimulated for 4 h with 100 nM SP, demonstrating by ELISA a pronounced diminution of IL-8 production in the N14Q/N18Q mutant cell line lacking both glycosylation sites. This reduction was significant when compared with cells containing the wild-type and single mutant receptors (α = 0.01, P < 0.001).

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