A Single Mutation of the Neurokinin-2 (NK2) Receptor Prevents Agonist-induced Desensitization

Abstract

Receptor activation and agonist-induced desensitization of the human neurokinin-2 (NK2) receptor expressed in Xenopus oocytes have been investigated. When neurokinin A (NKA) was applied repeatedly at 5-min intervals, the second and subsequent applications gave no responses. This desensitization was not observed with the specific agonists (Lys3, Gly8-R-gamma-lactam-Leu9)NKA(3-10) (GR64349) or (Nle10)-NKA(4-10). However, in the presence of the protein kinase inhibitor staurosporine, stimulation with GR64349 or (Nle10)-NKA(4-10) induced receptor desensitization. In contrast, the protein kinase C inhibitor Ro-31-8220 was not able to enhance GR64349-mediated desensitization. We created a mutation (F248S) in the third cytoplasmic loop of NK2 that impairs NKA-induced desensitization. In the presence of either staurosporine or Ro-31-8220, the mutant receptor was desensitized in response to NKA application but not to GR64349. Also, truncation mutants delta 62 and delta 87, lacking serine and threonine residues in the cytoplasmic COOH-terminal tail, were functionally active and were partially resistant to desensitization. These observations indicate that 1) there are different conformational requirements for NK2 receptor signalling and agonist-induced desensitization, 2) the third intracellular loop and the cytoplasmic tail of NK2 are functional domains important for agonist-induced desensitization, and 3) some agonists at the NK2 receptor cause much more desensitization than others and suggest that this might result from phosphorylation by receptor-specific kinases and other non-identified protein kinases.

The Neurokinin A Receptor Activates Calcium and cAMP Responses through Distinct Conformational States

“…We showed that two natural agonists, NKA and its truncated form, NKA-(4 -10) (24), differed in that NKA triggers rapidly desensitizing calcium responses, whereas repetitive NKA-(4 -10) administration did not alter cell responsiveness (23,25), supporting the conclusion that at least part of response desen-sitization to NKA was due to lack of receptor responsiveness.…”

“…It differs from NKA in that it repetitively activates intracellular calcium release both in oocytes (25) and HEK 293 cells (23) expressing the NK2R, and may be viewed as a "non-desensitizing" agonist with respect to calcium signaling. The fluorescent version of NKA-(4 -10) labeled with Texas Red used here, TR7-NKA-(4 -10), exhibits an apparent single step association to EGFP-NK2R that temporally correlates with the development of an intracellular calcium response (in the second to minute time range).…”

Probing the Structure and Function of the Tachykinin Neurokinin-2 Receptor through Biosynthetic Incorporation of Fluorescent Amino Acids at Specific Sites

“…The NK2 termination codon was TGA. Functional wild type NK2 expressed in Xenopus oocytes was characterized by inward currents of 1-4 A that appeared within 2-4 s after application of the natural agonist NKA and slowly decayed over 10 -20 s, corresponding to the activation of calcium-dependent chloride channel (24,30) (Fig. 2).…”

“…Saturation binding assays with PhCO-(␦-125 I-phenol-3-propionyl)-Orn-Ala-D-TrpPhe-D-Pro-Pro-Nle-NH 2 (0.1-10 nM) were performed as described previously (24). Nonspecific binding was determined in the presence of…”

“…Oocyte membrane fractions were prepared as described in Nemeth and Chollet (24). Electrophysiological recordings were carried out as described in Nemeth and Chollet (24).…”