Gαi RNA Antisense Expression Demonstrates the Exclusive Coupling of Peptide YY Receptors to Gi2 Proteins in Renal Proximal Tubule Cells

Thierry Voisin,Anne-Marie Lorinet,Jean-José Maoret,Alain Couvineau,Marc Laburthe

doi:10.1074/jbc.271.1.574

Abstract

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.

Highlights

Following its discovery in rat intestinal epithelial cells [1], the peptide YY (PYY)1 receptor has been characterized in dog adipocytes [2] and the proximal tubule PKSV-PCT cell line derived from kidneys of transgenic mice [3]
By studying receptor affinity and regulation of ligand binding by guanosine 5؅-O-(thiotriphosphate) (GTP␥S), inhibition of cAMP production and stimulation of cell growth in Cl.10 cell clones in which endogenous G␣i proteins were permanently down-regulated after transfection with antisense G␣i expression vectors, we provide evidence for the exclusive coupling of the PYY receptor to the Gi2 protein
Among the putative G protein subunits involved in the coupling of PYY receptors to biological events in Cl.10 cells, Western blot analysis indicated the presence of the Mr 39,000 G␣i2 subunit and the Mr 42,000 G␣i3 subunit but not G␣i1 subunits (Fig. 2)

Summary

Introduction

Following its discovery in rat intestinal epithelial cells [1], the peptide YY (PYY)1 receptor has been characterized in dog adipocytes [2] and the proximal tubule PKSV-PCT cell line derived from kidneys of transgenic mice [3]. The specific binding of 125I-Tyr0-PTH was identical in the two clones (not shown), demonstrating no alteration in PTH receptors in Cl.10/␣i2Ϫ cells consistent with the absence of changes in G␣s protein expression after transfection with G␣i2 antisense RNA (Fig. 2).

Results

Conclusion