EXPRESSION OF RAT NK-2 (NEUROKININ-A) RECEPTOR IN ESCHERICHIA-COLI

With the goal of obtaining sufficient functional protein for structura l analysis, rat neurokinin-2 receptor was produced in Escherichia coli by linking it to the periplasmic maltose-binding protein. As a first step, we present a biochemical and pharmacological investigation of th e recombinant receptor. Western-blots showed that the fusion protein w as associated with the membranes. The agonist [4,5-H-3-Leu(9)]neurokin in A and the NK-2 antagonist [H-3]SR48,968 bound to the receptor in a highly specific manner. Saturation binding of the [3H]agonist demonstr ated a single class of receptors (K-D = 10.5 nM, B-max = 2.5 pmol/mg p rotein). The [H-3]antag- onist bound with higher affinity to a larger receptor population (K-D = 0.2 nM, B-max = 7.2 pmol/mg protein). Compe tition of [H-3]agonist binding with other agonists demonstrated a pote ncy order of: neurokinin A > [Nle(10)]NKA(4-10) = [beta-Ala(8)]NKA(4-1 0) >> substance P >>> senktide. Against the [H-3]antagonist, agonists were only partially inhibitory. Selective NK-2 antagonists inhibited b inding of both [3H]ligands with an identical order of potency: SR48,96 8 >> R396 > MEN10,376, which is consistent with NK-2 receptor pharmaco logy in rat tissue.