Purification and reconstitution of a recombinant human neurokinin-1 receptor.

Recombinant human neurokinin-1 receptors expressed in insect cells have been purified to near homogeneity by sequential metal-chelating chromatography and gel filtration chromatography. The purified receptor consists of a single polypeptide with an apparent molecular weight of 50 kD as revealed by SDS gel electrophoresis, and exhibits a specific activity of 19 nmol of L-703,606 bound per mg of protein. Immunoblot experiments further confirm the identity of the stained protein band. The purified receptor binds the antagonist L-703,606 with an affinity similar to that of native human neurokinin-1 receptor, and binds the agonist substance P with an affinity similar to that of the low affinity state of uncoupled native receptor. The purified receptor can be reconstituted with membranes from uninfected insect cells, and the reconstitution results in an increased affinity for substance P, consistent with the reappearance of the high affinity state of the receptor for agonist in the presence of endogenous G proteins. These data indicate that the purified neurokinin-1 receptor is functional with respect to agonist and antagonist binding and G protein coupling.