G alpha i RNA antisense expression demonstrates the exclusive coupling of peptide YY receptors to G(i)2 proteins in renal proximal tubule cells.
A clone PKSV-PCT Cl.10 referred to as Cl.10 was selected from the PKSV-PCT renal proximal tubule cell line which expressed peptide YY (PYY) receptors (Voisin, T., Bens, M., Cluzeaud, F., Vandewalle, A., and Laburthe, M. (1993) J. Biol. Chem. 268, 20547-20554). In order to identify G(i) protein(s) coupled to PYY receptors, antisense G alpha i protein RNAs were expressed in Cl.10 cells by transfecting the pcDNA3 vector into which were inserted 39 bases of the 5'-noncoding region of G alpha i2 or G alpha i3 used as specific antisense templates. A Cl.10/alpha i2-clone was selected which displayed a drastic decrease (> 90%) of the expression of G alpha i2 without changes of G alpha i3, G alpha s, and G beta subunits (G alpha i1 is not present in Cl.10 cells) as evidenced by Western blots. When compared to untransfected cells, this clone exhibited: (i) an increase in the dissociation constant of PYY receptors (5.3 versus 0.6 nM) identical to that observed in pertussis toxin-treated untransfected cells; (ii) an absence of inhibition of 125I-PYY binding by guanosine 5'-O-(thiotriphosphate) (GTP gamma S); and (iii) the failure of PYY to inhibit cAMP levels and to stimulate [methyl-3H]thymidine incorporation into DNA. A clone was also selected which exhibited a specific decrease (> 80%) of G alpha i3 as compared to untransfected cells. The sensitivity to GTP gamma S and the dissociation constant of PYY receptors as well as PYY-mediated inhibition of cAMP were identical to those observed in untransfected cells. These findings support an exclusive coupling of PYY receptors to G alpha i2.
Unité de Recherche de Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santé et de la Recherche Médicale, INSERM U410, Faculté de Médecine Xavier Bichat, Paris, France.
T
T
Voisin
A M
AM
Lorinet
J J
JJ
Maoret
A
A
Couvineau
M
M
Laburthe
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002999:Clone Cells; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D016372:RNA, Antisense / Q000235:genetics*; D051381:Rats; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*; D013552:Swine
D016372:RNA, Antisense; D011964:Receptors, Gastrointestinal Hormone; C082172:peptide YY receptor; D019204:GTP-Binding Proteins