A substance P (neurokinin-1) receptor mutant carboxyl-terminally truncated to resemble a naturally occurring receptor isoform displays enhanced responsiveness and resistance to desensitization

Abstract

Two isoforms of the substance P (SP) receptor, differing in the length of the cytoplasmic carboxylterminus by Ϸ8 kDa, have been detected previously in rat salivary glands and other tissues. The binding and functional properties of these two isoforms have been investigated using full-length (407 amino acids) and carboxyl-terminally truncated ( These differences in responsiveness may be related to the observed differences in receptor desensitization. The truncated receptor, in contrast to the full-length receptor, does not undergo rapid and long-lasting desensitization. Cells possessing the short isoform of the SP receptor would thus be expected to exhibit a prolonged responsiveness.Substance P (SP), a member of the tachykinin family of peptides, is involved in many physiological processes, including exocrine gland secretion, vasodilation, pain transmission, and neurogenic inflammation (1). Molecular cloning established that the receptor for SP belongs to the G protein-coupled receptor family that possesses seven putative transmembrane domains (2, 3). Results from crosslinking and reconstitution studies have provided evidence that the SP receptor, also known as the neurokinin-1 receptor, couples to a subgroup of G proteins, Gq͞ 11 (4, 5). It has been shown in many tissues that SP activates phospholipase C ␤ (PLC ␤ ) which results in a transient increase in intracellular inositol 1,4,5-trisphosphate (IP 3 ) and cytosolic calcium concentration (6-9). The SP receptor, when stably expressed in Chinese hamster ovary (CHO) cells, also couples to phosphatidylinositol 4,5-bisphosphate (PIP 2 ) hydrolysis (10, 11).Biochemical studies in which the SP receptors in a membrane preparation of rat submaxillary glands were covalently labeled with a photoreactive SP analog (12, 13) have demonstrated the presence of two SP receptor isoforms that differ in the length of the cytoplasmic carboxyl-terminus by approximately 8 kDa. The photolabeling of the full-length SP receptor isoform (molecular mass, 46 kDa following deglycosylation) and the short isoform (molecular mass, 37 kDa following deglycosylation) are inhibited by SP with the same IC 50 (1 nM), indicating that both receptor isoforms bind SP with a similar affinity. A cDNA encoding a carboxyl-terminally truncated SP receptor with a calculated molecular weight of 35,797 has been cloned (14). When expressed in COS cells, this truncated receptor, in contrast to the receptor detected by photoaffinity labeling, has a binding affinity at least 10-fold less than that of the full-length receptor. Therefore, it is unlikely that the short receptor isoform identified in the rat salivary gland is derived from this spliced variant.To study the functional properties of the short SP receptor isoform identified in the rat salivary gland by photoaffinity labeling, a rat SP receptor mutant (324 amino acids; molecular weight, 37,360) carboxyl-terminally truncated to approximate the size of this short receptor isoform (13) was stably expressed in CHO cells (15). In this report, we have co...

Full-length and truncated neurokinin-1 receptor expression and function during monocyte/macrophage differentiation

“…Inositol 1,4,5-trisphosphate releases calcium from intracellular stores, and diacylglycerol increases intracellular calcium by opening voltage-gated Ca 2ϩ channels via PKC (60). The truncated receptor couples to calcium mobilization in transfected KNRK and CHO cells (33,34). These studies, however, are not inconsistent with our finding that the truncated NK-1R does not couple to calcium mobilization in monocytes͞macrophages (THP-1 cells).…”

“…Furthermore, weak or no responsiveness of the naturally truncated NK-1R to SP was also observed in another study examining the function of human full-length and truncated NK-1R (14). Based on these studies (14,(33)(34)(35) and our observation, we conclude that the domain between 311 and 324͞337 amino acid residues has a crucial role for NK-1R function(s) such as calcium mobilization. In the absence of this domain, SP will not induce calcium mobilization.…”

“…A distinct distribution of these two NK-1Rs has been observed in rat striatum, submaxillary glands, and patotid (32). The binding and signaling properties of the truncated receptor or mutants have been compared with those of the full-length receptor that is expressed in CHO cells, Xenopus oocytes, and KNRK cells (14,(33)(34)(35). Although the existence and expression of both the full-length and truncated NK-1R have been documented, the differences in their expression and physiological function, particularly in human monocytes͞macrophages, have not been studied.…”

Truncated neurokinin-1 receptor is increased in colonic epithelial cells from patients with colitis-associated cancer

“…In addition, Lai et al (26) showed that upon ligand binding, tr-NK-1R, which lacks a C-terminal tail that binds and activates G q , cannot initiate calcium influx or activate PKC and NF-κB; however, the truncated receptor can activate ERK in a delayed manner. The tr-NK-1R has also been reported to be resistant to desensitization (27,28). Not only does the truncated receptor lack C-terminal residues that are important for desensitization, it is also incapable of activating PKC (26), a critical component of the feedback inhibition loop that causes desensitization of the full-length receptor (28).…”

The proliferative and antiapoptotic effects of substance P are facilitated by formation of a β-arrestin-dependent scaffolding complex

“…A naturally occurring truncated variant of the NK1R (NK1R␦325) exhibits impaired SP-induced desensitization and endocytosis, possibly because of an inability to interact with ␤-arrestin (11,12). Therefore, we also compared the ability of wild type and truncated NK1R to activate ERK1͞2.…”