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 against the COOH-terminals of the alpha-subunits of Gi1/2, Gi3, and G(o), the binding and biological response of the Y2 receptor (Y2R) for peptide YY (PYY) was probed in SMS-KAN neuroblastoma cells. The specific binding of radiolabeled PYY exhibited a single apparent dissociation constant, KD = 76 pM for intact cells and KD = 906 pM for permeabilized cells. However, other data suggested existence of multiple receptor affinity states. A shift in KD and a decrease in apparent number of binding sites (Bmax) was observed in permeabilized cells when incubated with guanine nucleotides. By contrast, in membrane preparations guanine nucleotides induced only a decrease in Bmax. In intact cells, agonist exposure inhibited the intracellular accumulation of forskolin-stimulated cyclic AMP by 80% (IC50 = 420 nM) compared with 94% inhibition (IC50 = 380 nM) in permeabilized cells. In permeabilized cells, preincubation with antisera against alpha i1/2 and alpha i3 blocked the functional response of PYY, with anti-alpha i3 being the most potent; whereas anti-alpha o failed to affect the cyclic AMP levels. These results suggest that permeabilized SMS-KAN cells serve as a good model system for analysis of Y2R binding kinetics and functional response and that the Y2R interacts directly with several different GiS (but not G(o)).
Freitag
C
C
Department of Clinical Biochemistry, National University Hospital Rigshospitalet, Copenhagen, Denmark.
Svendsen
A B
AB
Feldthus
N
N
Løssl
K
K
Sheikh
S P
SP
D002463:Cell Membrane Permeability; D000242:Cyclic AMP / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D019894:Peptide YY; D010455:Peptides / Q000494:pharmacology; D037342:Pertussis Toxin; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*; D017476:Receptors, Neuropeptide Y / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D006150:Guanine Nucleotides; D007106:Immune Sera; D010455:Peptides; D011964:Receptors, Gastrointestinal Hormone; D017476:Receptors, Neuropeptide Y; D010566:Virulence Factors, Bordetella; C082172:peptide YY receptor; D019894:Peptide YY; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins