Differences in the length of the carboxyl terminus mediate functional properties of neurokinin-1 receptor.

The neurokinin-1 receptor (NK1R) has two naturally occurring forms that differ in the length of the carboxyl terminus: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. We examined whether there are differential signaling properties attributable to the carboxyl terminus of this receptor by using stably transfected human embryonic kidney (HEK293) cell lines that express either full-length or truncated NK1R. Substance P (SP) specifically triggered intracellular calcium increase in HEK293 cells expressing full-length NK1R but had no effect in the cells expressing the truncated NK1R. In addition, in cells expressing full-length NK1R, SP activated NF-kappaB and IL-8 mRNA expression, but in cells expressing the truncated NK1R, SP did not activate NF-kappaB, and it decreased IL-8 mRNA expression. In cells expressing full-length NK1R, SP stimulated phosphorylation of PKCdelta but inhibited phosphorylation of PKCdelta in cells expressing truncated NK1R. There are also differences in the timing of SP-induced ERK activation in cells expressing the two different forms of the receptor. Full-length NK1R activation of ERK was rapid (peak within 1-2 min), whereas truncated NK1R-mediated activation was slower (peak at 20-30 min). Thus, the carboxyl terminus of NK1R is the structural basis for differences in the functional properties of the full-length and truncated NK1R. These differences may provide important information toward the design of new NK1R receptor antagonists.

Functional disturbances in the striatum by region-specific ablation of NMDA receptors.

To study the role of NMDA receptors in dopamine signaling of the striatum, the brain area that receives glutamatergic inputs from various cortical areas and most dopaminergic inputs, we generated striatum-specific NMDA receptor-deficient mice. The mutant pups showed reduced food intake and retarded growth starting at the second postnatal week and died on approximately postnatal day 20 (P20). The time course of postnatal lethality is similar to that of compound mutant, double knockout of dopamine D1/D2 receptors, or genetically engineered dopamine-deficient mouse. In vivo electrophysiological recordings in the mutant pups showed that frequencies in the range of gamma oscillation were reduced in the striatal circuits. Moreover, the number of functional dopamine receptors in the striatum as measured by D1- and D2-binding experiments was greatly diminished in the mutants as compared with control animals. A consequence of diminished dopamine binding in the striatum manifested in an increase of locomotor activity. The administration of D1/D2 agonists paradoxically reduced the hyperactivity of the mutant mice as compared with an increase in locomotor activity in control mice. These results demonstrate that the NMDA receptor plays an essential role in the integration of dopamine signaling in the striatum and that is required in behavioral function.

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

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.