Parallel inactivation of Y2 receptor and G-proteins in CHO cells by pertussis toxin.
The Y(2) receptor for neuropeptide Y (NPY) interacts with pertussis toxin (PTX)-sensitive G-proteins, but little is known about interdependence of their levels and functions. We found that PTX reduces Y(2) receptors expressed in CHO cells in parallel to inactivation of Gi G-proteins, to loss of inhibition by Y(2) agonists of forskolin-stimulated adenylyl cyclase, and to decrease in the binding of GTP-gamma-S. These losses were attenuated by the endosome alkalinizer ammonium chloride. Affinity of the Y(2) receptor was not changed by PTX treatment. Prolonged treatment induced a large decrease of Y(2) receptor immunoreactivity (more than 70% in 48 h). The Gi(3) alpha-subunit immunoreactivity decreased slowly (about 46% in 48 h). There was a significant increase in Gq alpha immunoreactivity and in fraction of Y(2) binding sensitive to a Gq-selective antagonist. Possibly linked to that, the surface Y(2) sites and the internalization of the Y(2) receptor were less than 40% reduced. However, the abundant masked Y(2) sites were eliminated by the toxin, and could be mainly coupled to PTX-sensitive G-proteins.
Parker
S L
SL
Department of Pharmacology, University of Tennessee Health Science Center, Memphis, TN 38163, USA. slparker@utmem.edu <slparker@utmem.edu>
Parker
M S
MS
Sah
R
R
Sallee
F R
FR
Balasubramaniam
A
A
D000262:Adenylyl Cyclases / Q000378:metabolism; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D037342:Pertussis Toxin / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D017476:Receptors, Neuropeptide Y / Q000235:genetics / Q000378:metabolism*
D017476:Receptors, Neuropeptide Y; D000643:Ammonium Chloride; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases