Cell type-specific differences in the coupling of recombinant mGlu1alpha receptors to endogenous G protein sub-populations.
In this study the effects of cell background on the coupling of the type 1alpha metabotropic glutamate (mGlu1alpha) receptor to different G protein sub-populations by recombinant expression of this receptor subtype in baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cells have been investigated. Receptor-G protein interactions were assessed using [(35)S]GTPgammaS binding and subsequent Galpha subunit-specific immunoprecipitation. In a CHO cell line (CHO-lac-mGlu1alpha), where mGlu1alpha receptor expression is under inducible control, stimulation of membranes with the mGlu receptor agonist quisqualate resulted in an increase in specific [(35)S]GTPgammaS binding to G(q/11)alpha only, whereas in a BHK cell line (BHK-mGlu1alpha) agonist stimulation increased [(35)S]GTPgammaS binding to G(q/11)alpha and also to pertussis toxin (PTx)-sensitive G(i/o) proteins (assessed using G(i1/2)alpha- and G(i3/o)alpha-specific antibodies). These data are consistent with our previous observations of dual, antagonistic G(q/11)/G(i/o) regulation of phospholipase C (PLC) in BHK-mGlu1alpha cells, whereas no evidence was found for a G(i/o) modulation of PLC activity in the CHO-lac-mGlu1alpha cell line. PTx pre-treatment of either cell line had no effect on either the magnitude or the concentration-dependency of agonist-stimulated [(35)S]GTPgammaS-G(q/11)alpha binding, excluding the possibility that receptor-G(i/o) uncoupling can unmask an increase in receptor-G(q/11) interaction. mGlu1alpha receptor expression per se had little effect on Galpha protein expression levels in either CHO or BHK cell lines, with the possible exception of a small, but consistent increase in G(o)alpha expression in BHK-mGlu1alpha cells compared to the vector-transfected control cell line (BHK-570). Semi-quantitative assessment of mGlu1alpha receptor immunoreactivity and [(3)H]quisqualate saturation binding analysis demonstrated a ca 10-fold higher mGlu1alpha receptor content in BHK cells. Whether the higher receptor expression level in BHK-mGlu1alpha cells underlies the additional G(i/o) coupling observed in this cell line, or additional factors contribute to the phenomenon are discussed.
Selkirk
J V
JV
Department of Cell Physiology and Pharmacology, Maurice Shock Medical Sciences Building, University of Leicester, University Road, Leicester LE1 9HN, UK. jvs2@le.ac.uk
Price
G W
GW
Nahorski
S R
SR
Challiss
R A
RA
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects; D002478:Cells, Cultured; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007295:Inositol Phosphates / Q000494:pharmacology; D009474:Neurons / Q000378:metabolism*; D037342:Pertussis Toxin; D011233:Precipitin Tests; D016318:Quisqualic Acid / Q000494:pharmacology; D018094:Receptors, Metabotropic Glutamate / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007295:Inositol Phosphates; D018094:Receptors, Metabotropic Glutamate; D010566:Virulence Factors, Bordetella; C104077:metabotropic glutamate receptor type 1; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D016318:Quisqualic Acid; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins