Characterization of a metabotropic glutamate receptor type 5-green fluorescent protein chimera (mGluR5-GFP): pharmacology, surface expression, and differential effects of Homer-1a and Homer-1c.
Metabotropic glutamate receptor 5 (mGluR5) can modulate synaptic transmission by increasing intracellular Ca2+ and it plays a role in several forms of synaptic plasticity. We have constructed a fusion of human mGluR5 and green fluorescent protein (mGluR5-GFP). Expression of mGluR5-GFP in clonal cell lines yielded a functional fluorescent receptor with pharmacological profiles similar to wild-type mGluR5. mGluR5-GFP coimmunoprecipitated with Homer-1c, indicating that addition of GFP to the C-terminal did not prevent Homer binding. Coexpression of wild-type mGluR5 or mGluR5-GFP with Homer 1c, but not Homer-1a, resulted in reduced receptor surface localization and the formation of intracellular clusters. Neither Homer-1a nor Homer-1c had any effect on mGluR1 or mGluR1-GFP distribution. mGluR5-GFP expressed alone or in combination with Homer-1a formed dimers in HEK cells. Coexpression with Homer-1c, however, prevented mGluR5-GFP dimerization. Neither Homer altered the agonist profiles of mGluR5 or mGluR5-GFP. These data indicate that the functional expression of mGluR5 is regulated by Homer-1c and demonstrate that mGluR5-GFP provides a useful tool to study the molecular pharmacology and cell biology of mGluRs in real-time.
Coutinho
V
V
MRC Centre for Synaptic Plasticity, University Walk, Bristol, BS8 1TD, United Kingdom.
Kavanagh
I
I
Sugiyama
H
H
Tones
M A
MA
Henley
J M
JM
D000818:Animals; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis / Q000502:physiology*; D006224:Cricetinae; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D000072233:Homer Scaffolding Proteins; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000235:genetics*; D009479:Neuropeptides / Q000096:biosynthesis / Q000502:physiology*; D011233:Precipitin Tests; D011725:Pyridines / Q000494:pharmacology; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014162:Transfection / Q000379:methods
D002352:Carrier Proteins; D018691:Excitatory Amino Acid Antagonists; C578935:GRM5 protein, human; D000072233:Homer Scaffolding Proteins; D008164:Luminescent Proteins; D009479:Neuropeptides; D011725:Pyridines; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C121465:6-methyl-2-(phenylethynyl)pyridine