Characterization of the cloned neurokinin A receptor transfected in murine fibroblasts

Abstract

The neurokinins comprise a family of peptides with similar structure and physiological effects. Several limitations arise when investigating their biochemical and pharmacological properties. Because neurokinins contain similar amino acids in their carboxyl terminus, radiolabeled neurokinins and biosynthesized antibodies may lack the specificity to distinguish between the various neurokinin receptors. Tissue preparations used to characterize their pharmacological responses often have several types of neurokinin receptors, different receptor densities, or signal transduction mechanisms. These factors can complicate the interpretation of results from functional assays. Developing a cell line that expresses a high density of one type of neurokinin receptor with similar biochemical and pharmacological characteristics as the endogenous receptor may overcome these limitations. To facilitate this aim, the neurokinin-2 receptor (NK-2) was expressed by transfecting a bovine stomach cDNA that encoded for the NK-2 receptor into the murine fibroblast B82 L cell line. This de novo protein displayed high affinity and selectivity for neurokinin A (NKA) in competitive radioligand binding assays and was functionally coupled to phosphatidylinositide 4,5-bisphosphate (PIP₂) hydrolysis and intracellular calcium mobilization. The expressed receptor also exhibited a time dependent, homologous desensitization of PIP₂ hydrolysis. The rank order of affinity in competitive [¹²⁵I]NKA binding and potency for accumulation of [³H] inositol monophosphates ([³H]IP₁) in transfected B82 fibroblasts was NKA > substance P > senktide which classified the expressed protein as an NK-2 receptor. The expressed NK-2 receptors in B82 fibroblasts and the endogenous NK-2 receptors in bovine stomach had similar pharmacological properties. This implies that the in vitro expression system produced a protein whose pharmacological and biochemical characteristics resemble the endogenous receptor. The antagonists MEN 10207 and MEN 10208 blocked NKA-induced PIP₂ hydrolysis. In addition, the different pA₂ values of these peptides in transfected B82 cells, rabbit pulmonary artery, and hamster trachea of these peptides suggest NK-2 receptor heterogeneity in these tissues. The availability of a cell line expressing a homogeneous population of NK-2 receptors that mimics the phramacological characteristics of the endogenous receptor allows closer examination of ligand-receptor-effector interactions in a controlled environment. The novel reporting of an NK-2 receptor subtype and desensitization may provide a mechanism for intervention of physiological responses mediated by this receptor subtype.