COUPLING OF THE HUMAN Y2 RECEPTOR FOR NEUROPEPTIDE-Y AND PEPTIDE YY TO GUANINE-NUCLEOTIDE INHIBITORY PROTEINS IN PERMEABILIZED SMS-KAN CELLS

Using guanine nucleotides, pertussis toxin, and specific antisera agai nst the COOH-terminals of the alpha-subunits of G(i1/2), G(i3), and G( o), the binding and biological response of the Y2 receptor (Y2R) for p eptide YY (PYY) was probed in SMS-KAN neuroblastoma cells. The specifi c binding of radiolabeled PW exhibited a single apparent dissociation constant, K-D = 76 pM for intact cells and K-D = 906 pM for permeabili zed cells. However, other data suggested existence of multiple recepto r affinity states. A shift in K-D and a decrease in apparent number of binding sites (B-max) was observed in permeabilized cells when incuba ted with guanine nucleotides. By contrast, in membrane preparations gu anine nucleotides induced only a decrease in B-max. In intact cells, a gonist exposure inhibited the intracellular accumulation of forskolin- stimulated cyclic AMP by 80% (IC50 = 420 nM) compared with 94% inhibit ion (IC50 = 380 nM) in permeabilized cells. In permeabilized cells, pr eincubation with antisera against alpha(i1/2) and alpha(i3) blocked th e functional response of PYY, with anti-alpha(i3) being the most poten t; whereas anti-alpha(o) failed to affect the cyclic AMP levels. These results suggest that permeabilized SMS-KAN cells serve as a good mode l system for analysis of Y2R binding kinetics and functional response and that the Y2R interacts directly with several different G(i)s (but not G(o)).