Partial knockdown of G alpha i2 protein is sufficient to abolish the coupling of PYY receptors to biological response in renal proximal tubule cells.
Peptide YY (PYY)-preferring receptors are expressed in the renal proximal tubule cell clone Cl.10 isolated from the PKSV-PCT cell line. They mediate PYY-inhibited cAMP production through coupling with pertussis-sensitive Gi proteins. Previous G alpha i RNA antisense experiments demonstrated the exclusive coupling of the PYY receptor to the Gi2 protein. Here we characterized a clone stably expressing G alpha i2 antisense RNA which exhibited only a partial decrease in G alpha i2 content (#60%) as estimated by Western blot. When compared to control Cl.10 cells, this clone, referred to as Cl.10(t), exhibited: (i) an increase in the dissociation constant of PYY receptors (6.42 vs 0.63 nM); (ii) a complete absence of inhibition of [125I]PYY binding by GTP gamma S and GTP; (iii) the failure of PYY to inhibit basal and forskolin-stimulated cAMP levels; (iv) the failure of PYY to stimulate [35S]GTP gamma S binding to membranes. These findings show that partial knockdown of G alpha i2 expression in Cl.10 cells completely abolish the coupling of PYY receptors to biological response.
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Unité de Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santé et de la Recherche Médicale, INSERM U410, Faculté de Médecine X. Bichat, Paris, France.
Lorinet
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Laburthe
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D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002999:Clone Cells; D000242:Cyclic AMP / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D015151:Immunoblotting; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D019894:Peptide YY; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*; D011964:Receptors, Gastrointestinal Hormone / Q000187:drug effects / Q000502:physiology*; D013552:Swine; D014162:Transfection
D046911:Macromolecular Substances; D016376:Oligonucleotides, Antisense; D010455:Peptides; D011964:Receptors, Gastrointestinal Hormone; C082172:peptide YY receptor; D019894:Peptide YY; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D019204:GTP-Binding Proteins