Expression of rat NK-2 (neurokinin A) receptor in E. coli.
With the goal of obtaining sufficient functional protein for structural 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 the recombinant receptor. Western-blots showed that the fusion protein was associated with the membranes. The agonist [4,5-3H-Leu9]neurokinin A and the NK-2 antagonist [3H]SR48,968 bound to the receptor in a highly specific manner. Saturation binding of the [3H]agonist demonstrated a single class of receptors (KD = 10.5 nM, Bmax = 2.5 pmol/mg protein). The [3H]antagonist bound with higher affinity to a larger receptor population (KD = 0.2 nM, Bmax = 7.2 pmol/mg protein). Competition of [3H]agonist binding with other agonists demonstrated a potency order of: neurokinin A > [Nle10]NKA(4-10) = [beta-Ala8]NKA(4-10) >> substance P >>> senktide Against the [3H]antagonist, agonists were only partially inhibitory. Selective NK-2 antagonists inhibited binding of both [3H]ligands with an identical order of potency: SR48,968 >> R396 > MEN10,376, which is consistent with NK-2 receptor pharmacology in rat tissue.
Grisshammer
R
R
Cambridge Centre for Protein Engineering/MRC Centre, UK.
Little
J
J
Aharony
D
D
D018528:ATP-Binding Cassette Transporters; D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000096:biosynthesis; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D015870:Gene Expression; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D015288:Neurokinin A / Q000378:metabolism*; D033902:Periplasmic Binding Proteins; D011869:Radioligand Assay; D051381:Rats; D018041:Receptors, Neurokinin-2 / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D014316:Tritium
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D018041:Receptors, Neurokinin-2; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C461599:maltose transport system, E coli; D014316:Tritium; D008320:Maltose; D015288:Neurokinin A