I agree. Stable are good in 293 and transient in COS-7.
Idf the receptor is getting internalized when you culture the cells, check for what is called Tonic internalization. Some receptors fo it. I don't know what happens to yours. You can keep the cells on ice.
You can check the PAR-1 and thromboxane receptors

Could you please write your best protocol for a successful transient and stable transfection leading to accuratelty folded cell surface expressions?. I work with this odorant GPCR and I need to make monoclonal antibody against the cell surface protein. In most cases I only get internal exppression.[/quote]

GPCR molecules expressed in bacteria can be used later for monoclonal antibody production. In the paper below the method of expression in E.coli has been desribed:
SCH-202676: An Allosteric Modulator of Both Agonist and Antagonist Binding to G Protein-Coupled Receptors Ahmad B. Fawzi, Douglas Macdonald,1 Lawrence L. Benbow, April Smith-Torhan, Hongtao Zhang, Blair C. Weig, Ginny Ho, Deen Tulshian, Maurine E. Linder, and Michael P. Graziano

Methods of expression

Expression of Human 2-Adrenergic Receptor in DH5 Bacterial Cells. The 2-adrenergic receptor was expressed in Escherichia coli by a modification of methods described previously by Marullo et al. (1988) and Freissmuth et al. (1991). The human 2-adrenergic receptor (cDNA obtained from Dr. R. Lefkowitz, GenBank accession number 4501968) was amplified using polymerase chain reaction primers designed to incorporate an EcoRI site at the 5'-end, and SalI at the 3'-end (upper primer: 5'-CTTGAATTCGGGCAACCCGGGAACGG-3', lower primer: 5'-TCTGTCGACTTACAGCAGTGAGTCATT-3', respectively). After digestion with the corresponding restriction enzymes, the polymerase chain reaction product was ligated into a pFLAG-1 vector (Eastman Kodak Co., Rochester, NY). The nucleotide sequence of the pFLAG-1 2-adrenergic receptor cDNA was verified using the dRhodamine Terminator Cycle Sequencing Reaction system (PE Biosystems, Foster City, CA) and analyzed on an ABI PRISM 377 automated DNA sequencer (PE/ABI, Foster City, CA). Transformation of the purified plasmid into E. coli strain DH5 was performed using the standard commercial protocol provided with the DH5 competent cells (Life Technologies, Gaithersburg, MD). The pFLAG/2-adrenergic receptor-positive DH5 transformants were cultured at 37°C in ampicillin-containing (100 g/ml) Luria broth culture to an optical density of 500 (= 600 nm) at which point 0.5 mM isopropylthio--D-galactoside was added. After additional incubation for 2.5 h at 23°C, membranes were isolated as described previously (Stanasila et al., 1999). Membrane pellets were resuspended in 1 ml of cold 50 mM Tris-HCl, pH 7.4, containing 10% glycerol and 1% BSA. Aliquots were frozen in liquid nitrogen and stored at 80°C. Protein determinations were made before the BSA addition using the micro bicinchoninic acid assay (BCA; Pierce, Rockford, IL). Competition binding of [125I]iodocyanopindolol to 50 g of pFLAG-1/2-adrenergic receptor DH5 membranes in 500 l of buffer containing 75 mM Tris-HCl, pH 7.4, 12.5 mM MgCl2, 2 mM EDTA, and protease inhibitors (Complete+, EDTA; Boehringer Mannheim) was performed as described before (Perkin Elmer Life Sciences, Norwalk, CT).

GPCR molecules expressed in bacteria can be used later for monoclonal antibody production. In the paper below the method of expression in E.coli has been desribed:
SCH-202676: An Allosteric Modulator of Both Agonist and Antagonist Binding to G Protein-Coupled Receptors Ahmad B. Fawzi, Douglas Macdonald,1 Lawrence L. Benbow, April Smith-Torhan, Hongtao Zhang, Blair C. Weig, Ginny Ho, Deen Tulshian, Maurine E. Linder, and Michael P. Graziano

Methods of expression

Expression of Human 2-Adrenergic Receptor in DH5 Bacterial Cells. The 2-adrenergic receptor was expressed in Escherichia coli by a modification of methods described previously by Marullo et al. (1988) and Freissmuth et al. (1991). The human 2-adrenergic receptor (cDNA obtained from Dr. R. Lefkowitz, GenBank accession number 4501968) was amplified using polymerase chain reaction primers designed to incorporate an EcoRI site at the 5'-end, and SalI at the 3'-end (upper primer: 5'-CTTGAATTCGGGCAACCCGGGAACGG-3', lower primer: 5'-TCTGTCGACTTACAGCAGTGAGTCATT-3', respectively). After digestion with the corresponding restriction enzymes, the polymerase chain reaction product was ligated into a pFLAG-1 vector (Eastman Kodak Co., Rochester, NY). The nucleotide sequence of the pFLAG-1 2-adrenergic receptor cDNA was verified using the dRhodamine Terminator Cycle Sequencing Reaction system (PE Biosystems, Foster City, CA) and analyzed on an ABI PRISM 377 automated DNA sequencer (PE/ABI, Foster City, CA). Transformation of the purified plasmid into E. coli strain DH5 was performed using the standard commercial protocol provided with the DH5 competent cells (Life Technologies, Gaithersburg, MD). The pFLAG/2-adrenergic receptor-positive DH5 transformants were cultured at 37°C in ampicillin-containing (100 g/ml) Luria broth culture to an optical density of 500 (= 600 nm) at which point 0.5 mM isopropylthio--D-galactoside was added. After additional incubation for 2.5 h at 23°C, membranes were isolated as described previously (Stanasila et al., 1999). Membrane pellets were resuspended in 1 ml of cold 50 mM Tris-HCl, pH 7.4, containing 10% glycerol and 1% BSA. Aliquots were frozen in liquid nitrogen and stored at 80°C. Protein determinations were made before the BSA addition using the micro bicinchoninic acid assay (BCA; Pierce, Rockford, IL). Competition binding of [125I]iodocyanopindolol to 50 g of pFLAG-1/2-adrenergic receptor DH5 membranes in 500 l of buffer containing 75 mM Tris-HCl, pH 7.4, 12.5 mM MgCl2, 2 mM EDTA, and protease inhibitors (Complete+, EDTA; Boehringer Mannheim) was performed as described before (Perkin Elmer Life Sciences, Norwalk, CT).



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Posted on Yesterday, 3:14 pm

Dose anyone know more about the coupling of GPCR and G proteins, as we know, the intracellular regions of GPCRs are mainly decide the coupling specificity to different G proteins,
I want to know is there existing some features in GPCRs coupling to different G proteins, how could they identify different G proteins and couple with it.

I've been working with ORs for some time now. Our lab is using xenopus oocytes. The key things we've noted as being important are the n'term rhodopsin tag, and occassionally (but not very often) the accessory proteins from hiro matsunami's lab. We've done minimal expression studies w/ receptors like b2-AR and aren't impressed. Goodluck- until people try these receptors themselves, I dont think they realize how tricky they can be

A nice review of GPCR trafficking and folding can be found here

Biochimica et Biophysica Acta (BBA) - Biomembranes
Volume 1768, Issue 4, April 2007, Pages 853-870

Entitled the

Regulation of G protein-coupled receptor export trafficking

Abstract

G protein-coupled receptors (GPCRs) constitute a superfamily of cell-surface receptors which share a common topology of seven transmembrane domains and modulate a variety of cell functions through coupling to heterotrimeric G proteins by responding to a vast array of stimuli. The magnitude of cellular response elicited by a given signal is dictated by the level of GPCR expression at the plasma membrane, which is the balance of elaborately regulated endocytic and exocytic trafficking. This review will cover recent advances in understanding the molecular mechanism underlying anterograde transport of the newly synthesized GPCRs from the endoplasmic reticulum (ER) through the Golgi to the plasma membrane. We will focus on recently identified motifs involved in GPCR exit from the ER and the Golgi, GPCR folding in the ER and the rescue of misfolded receptors from within, GPCR-interacting proteins that modulate receptor cell-surface targeting, pathways that mediate GPCR traffic, and the functional role of export in controlling GPCR signaling.


In particular there are sections on the Rescue of misfolded GPCRs by chemical and pharmacological chaperones or by lowering the culture temperature

You could try looking at this article and others by Dr. Luetje, although a lot of his work is in Xenopus oocytes, it could provide leads to answer your problem with expression of functional receptors.  he does mention use of chaperone proteins with some receptors..

J Neurochem. 2006 Jun;97(5):1506-18. Epub 2006 Apr 5.

Functional analysis of a mammalian odorant receptor subfamily.

Abaffy T, Matsunami H, Luetje CW.

Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami, Miami, Florida 33101, USA.