PURIFICATION AND RECONSTITUTION OF A RECOMBINANT HUMAN NEUROKININ-1 RECEPTOR

Recombinant human neurokinin-1 receptors expressed in insect cells hav e been purified to near homogeneity by sequential metal-chelating chro matography and gel filtration chromatography. The purified receptor co nsists of a single polypeptide with an apparent molecular weight of 50 kD as revealed by SDS gel electrophoresis, and exhibits a specific ac tivity of 19 nmol of L-703,606 bound per mg of protein. Immunoblot exp eriments further confirm the identity of the stained protein band. The purified receptor binds the antagonist L-703,606 with an affinity sim ilar to that of native human neurokinin-1 receptor, and binds the agon ist substance P with an affinity similar to that of the low affinity s tate of uncoupled native receptor. The purified receptor can be recons tituted with membranes from uninfected insect cells, and the reconstit ution results in an increased affinity for substance P, consistent wit h the reappearance of the high affinity state of the receptor for agon ist in the presence of endogenous G proteins. These data indicate that the purified neurokinin-1 receptor is functional with respect to agon ist and antagonist binding and G protein coupling.