GPCR

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Sandy
Sandy's picture
GPCR

G protein coupled receptors are very interesting cell surface molecules. I have done several experiments trying to express the GPCR target we are working at in my lab. Cell lines I have chosen cannot express this molecule easily and the expression is lost or internalized quickly. Has anybody tried to express this molecule, which cell lines?

DD
DD's picture
GPCRs belong to a very big

GPCRs belong to a very big family of 7 transmemebrane domain. The recombinant cells expressing GPCR loose their expression very quickly. The best cells to use to my knowledge are: 293T transient cells. They cannot be stabilized but they express the protein very high for a short period time.

vanishing
vanishing's picture
which GPCR are you trying to

which GPCR are you trying to express?

Sandy
Sandy's picture
vanishing wrote:which GPCR

vanishing wrote:

which GPCR are you trying to express?

The GPCR molecule I am trying to express is an odorant receptor expressed on olfactory epithelial cells. It belongs to the class A of GPCRs based on Transmembrane homology. With the amino terminus outside and 7 transmembrane domains and the carboxy terminus inside.
Thanks for helping.

Maura Matus-Ortega
Maura Matus-Ortega's picture
Hi! I was working whith the

Hi! I was working whith the Mu and Delta opioid receptors that are CPCR's then they were expresed by CHO's cells.
There were no problems.

omid
omid's picture
MMatus wrote:Hi! I was

MMatus wrote:

Hi! I was working whith the Mu and Delta opioid receptors that are CPCR's then they were expresed by CHO's cells.
There were no problems.

Most of people have problems expressing GPCRs. Did you ever used a transiant expression? I beleive the CHO cells are for stable transfection?

vanishing
vanishing's picture
Is there an antagonist to the

Is there an antagonist to the receptor? Treating the cells in culture could inhibit internalization...

You could also think of trying an inducible expression system? (Tet-Off)?

Maura Matus-Ortega
Maura Matus-Ortega's picture
Hi again, we used the mu and

Hi again, we used the mu and delta GPCRs cloned at pCDNA3 plasmid, then it was transfected at CHOS cells. We have done some bioassays with those cultures.

stevenson
stevenson's picture
In general GPCRs are

In general GPCRs are relatively not that difficult to express. I've been doing GPCR expression for more than ten years now and the more common mammalian cell lines I've used have been HEK293 cells, CHO-K1 for stable expression and COS-7 cells for transient. An optimized transfection protocol is key to a good protein expression system. It's also good to know how your ligand binds to the receptor in case there's a temperature/mechanical variable that you have to look out for. For large scale protein expression/production, the Sf9 insect cell line using baculoviruses has been particularly valuable for GPCR expression-not to mention the scalability! Good luck!

mottagui
mottagui's picture
May be I missed the point,

May be I missed the point, but we have expressed numerous GPCRs in several different cell lines (HeLa, SK-NMc, HL60, Jutkat, Hek293 and U937) and they mostly expressed in a masureble manner (Ct= 29-32)
We clone them on pGBasic and are quite OK with working with them. But then , I have recently ventured into this area of biology, so some of you know much more than me.
Enlighten me!!

M

R
R's picture
Sandy wrote:G protein coupled

Sandy wrote:

G protein coupled receptors are very interesting cell surface molecules. I have done several experiments trying to express the GPCR target we are working at in my lab. Cell lines I have chosen cannot express this molecule easily and the expression is lost or internalized quickly. Has anybody tried to express this molecule, which cell lines?

Have you checked eg:
1] J Biol Chem. 2005 Mar 25;280(12):11807-15.
2] Proc Natl Acad Sci U S A. 2004 Sep 14;101(37):13672-6.

Where they [1] set up an assay system for odorant GPCRs in HeLa cells; or [2] improve cell surface delivery of odorant GPCR by coexpressing the receptor of interest with the beta2 adrenergic receptor.

good luck
R

Sandy
Sandy's picture
stevenson wrote:In general

stevenson wrote:

In general GPCRs are relatively not that difficult to express. I've been doing GPCR expression for more than ten years now and the more common mammalian cell lines I've used have been HEK293 cells, CHO-K1 for stable expression and COS-7 cells for transient. An optimized transfection protocol is key to a good protein expression system. It's also good to know how your ligand binds to the receptor in case there's a temperature/mechanical variable that you have to look out for. For large scale protein expression/production, the Sf9 insect cell line using baculoviruses has been particularly valuable for GPCR expression-not to mention the scalability! Good luck!

Thank you so much for your valuable input. We have tried many cell lines except the CHO-K1, we will try this cell line for the stable expression.

magali
magali's picture
Odorant receptors are often

Odorant receptors are often difficult to express, probably because they need special chaperones for proper folding. Why don't you look at these two papers:

Alexander A. Gimelbrant, Shannon L. Haley, and Timothy S. McClintock
Olfactory Receptor Trafficking Involves Conserved Regulatory Steps
J. Biol. Chem., Vol. 276, Issue 10, 7285-7290, March 9, 2001
(a special cell line that allows maturation of some of these receptors), and
J. Minic, M.-A. Persuy, E. Godel, J. Aioun, I. Connerton, R. Salesse, and E. Pajot-Augy
Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening
FEBS J., January 15, 2005; 272(2): 524 - 537.
(summarizes tricks that have worked with some difficult-to-express odorant receptors)

roudi
roudi's picture
magali wrote:Odorant

magali wrote:

Odorant receptors are often difficult to express, probably because they need special chaperones for proper folding. Why don't you look at these two papers:

Alexander A. Gimelbrant, Shannon L. Haley, and Timothy S. McClintock
Olfactory Receptor Trafficking Involves Conserved Regulatory Steps
J. Biol. Chem., Vol. 276, Issue 10, 7285-7290, March 9, 2001
(a special cell line that allows maturation of some of these receptors), and
J. Minic, M.-A. Persuy, E. Godel, J. Aioun, I. Connerton, R. Salesse, and E. Pajot-Augy
Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening
FEBS J., January 15, 2005; 272(2): 524 - 537.
(summarizes tricks that have worked with some difficult-to-express odorant receptors)

In the recent work of Sayako et al. published in the journal of Neurochemistry, volume 90 issue 6 page 1453, September 2004, the importance of N-terminal glycosylation has been shown to be critical for OR expression and membrane trafficking. The C-terminal portion plays a role in defining proper conformation which specifies the G protain selectivity of the OR.
Disruption of the N-terminal glycosylation site completely impairs its membrane trafficking. Functional expression of the mOR-EG was geatly enhanced by addition of extra-N terminal glycosylation sequences.

Sandy
Sandy's picture
stevenson wrote:In general

stevenson wrote:

In general GPCRs are relatively not that difficult to express. I've been doing GPCR expression for more than ten years now and the more common mammalian cell lines I've used have been HEK293 cells, CHO-K1 for stable expression and COS-7 cells for transient. An optimized transfection protocol is key to a good protein expression system. It's also good to know how your ligand binds to the receptor in case there's a temperature/mechanical variable that you have to look out for. For large scale protein expression/production, the Sf9 insect cell line using baculoviruses has been particularly valuable for GPCR
expression-not to mention the scalability! Good luck!

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.

tash
tash's picture
Quote:In most cases I only

Quote:

In most cases I only get internal exppression.

Some people use a signal sequence to enhance initial trafficking of your receptor to the cell surface. I think the sequence is cleaved from the protein during the protein synthesis process, thus downstream intracellular trafficking of the receptor should not be affected. An example is the pplss (preprolactin signal sequence).

You'd have to engineer such a sequence into your gene construct, however, and I expect that final cell surface receptor expression would be dependent on such things as the rate of new receptor production (based on your promoter) and the rate of constitutive / media-component triggered intracellular trafficking.

Nancy
Nancy's picture
I agree. Stable are good in

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

roudi
roudi's picture
Sandy wrote:stevenson wrote

Sandy wrote:

stevenson wrote:
In general GPCRs are relatively not that difficult to express. I've been doing GPCR expression for more than ten years now and the more common mammalian cell lines I've used have been HEK293 cells, CHO-K1 for stable expression and COS-7 cells for transient. An optimized transfection protocol is key to a good protein expression system. It's also good to know how your ligand binds to the receptor in case there's a temperature/mechanical variable that you have to look out for. For large scale protein expression/production, the Sf9 insect cell line using baculoviruses has been particularly valuable for GPCR expression-not to mention the scalability! Good luck!

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.

Not all the GPCRs are expressed in mammalian cell lines. They can be expressed in bacteria with great success:

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.
below is the protocol:
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).

roudi
roudi's picture
Could you please write your

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.

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).

Soudabeh
Soudabeh's picture
GPCR molecules expressed in

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

Guan Cuiping
Guan Cuiping's picture
Dose anyone know more about

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.

SRep
SRep's picture
I've been working with ORs

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

montgomj
montgomj's picture
A nice review of GPCR

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

Bluejay
Bluejay's picture
You could try looking at this

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.