CHEMICAL CROSS-LINKING OF THE SUBSTANCE-P (NK-1) RECEPTOR TO THE ALPHA-SUBUNITS OF THE G-PROTEINS G(Q) AND G(11)

We have previously shown that the high-affinity binding of substance P (SP) to its receptor is dependent on an interaction with a PTX-insens itive G protein. This G protein couples SP receptor activation to stim ulation of its effector, phospholipase C. In this study, we combined p hotoaffinity labeling, chemical cross-linking techniques, and immunolo gical characterization using sequence-specific antibody probes to iden tify G proteins that couple to the SP receptor. First we covalently la beled the SP receptor present on rat submaxillary gland membranes with a radioiodinated photoreactive derivative of SP: p-benzoyl-L-phenylal anine(8)-substance P (I-125-[Bpa(8)]SP). Photoincorporation of this SP derivative was susceptible to guanine nucleotide inhibition, indicati ng that the receptor was coupled to its G protein during labeling, We then used a chemical cross-linking agent to covalently link the photoa ffinity labeled SP receptor and its associated G protein. Cross-linkin g generated a 96 kDa product, formation of which was prevented by the addition of a guanine nucleotide, but not an adenine nucleotide, follo wing photolabeling, but prior to cross-linking. Furthermore, the 96 kD a cross-linked complex was absent in membranes which had been depleted of G proteins by treatment with alkaline buffer prior to addition of the cross-linking agent. Reductive cleavage of the cross-link in the i solated 96 kDa complex yields two products: the 53 kDa SP receptor and a 42 kDa protein identified by immunoblot analysis as either G alpha( q) or G alpha(11). Antisera against a common sequence within G alpha(s ), G alpha(i), and G alpha(o) showed no immunoreactivity to the comple x or its cleavage products, These results provide the first direct evi dence of specific interaction between photoaffinity labeled SP recepto r and the alpha subunits of G(q) and G(11), members of a family of G p roteins known to be associated with pertussis toxin-insensitive phosph olipase C activation.