Hi Argha Mario,

Keeping in mind your request the protocol can be understood for biology students, I would like to recommend to take as a prototype the GPR6/ It is a constitutively active Gs-coupled receptor that can signal from intracellular compartments. GPR6 belongs to a family of constitutively active Gs-coupled G protein coupled receptors (GPCRs) that also includes GPR3 and GPR12.

There are the following four methods of detecting cell surface expression of GPCRs:

1. Cell surface biotinylation

Biotinylation of cell surface proteins with amine-reactive compounds is a commonly used technique to label cell surface proteins which can then be separated and quantified. Sulfo NHS-SS biotin, which is frequently used for this purpose, is a membrane impermeant compound that reacts with amino groups of extracellular lysine side chains in proteins. We used sulfo NHS-SS biotin to label cell surface proteins and measure the relative abundance of pHluorin-tagged GPR6 (pHGPR6) and D1R (pHD1) on the cell surface. As will be evident later in this chapter, the pHluorin tag at the N-terminus will be used to measure cell surface expression of GPR6 in different assays. Super ecliptic pHluorin is a pH sensitive variant of GFP (pKa ~ 7.1) which can easily be detected by commercially available GFP antibodies.

2. Susceptibility to chymotrypsin treatment

Chymotrypsin, an endoprotease secreted by pancreas, cleaves proteins at aromatic amino acid residues (tyrosine, tryptophan or phenylalanine). Brief exposure to this enzyme can be used to cleave cell surface proteins without dislodging cells from the culture dish. This enzyme is relatively large (~ 25 kDa) and does not have access to intracellular proteins of intact cells. We found that a pHlourin tag at the N-terminus of cell surface GPCRs is cleaved within a few seconds of exposure to chymotrypsin. The cleavage of pHluorin tag could thus be used as a read-out for detection and quantification of receptors on the cell surface.

3. Imaging based assays for cell surface expression

Although the data presented above show that GPR6 is located in the intracellular compartments of HEK293 cells, it is important to determine the expression pattern of GPR6 in neurons where it is normally expressed. It is conceivable that a missing subunit or chaperone of GPR6 is required for its surface expression in heterologous expression systems. We next assessed expression of GPR6 in neurons that are likely to have these putative GPR6 associated proteins. However, low transfection efficiency of GPR6 in neurons precluded us from using either of the above two methods. We resorted to imaging based analysis of surface expression in individual cells that are transfected with pHGPR6 or pHD1. This assay takes advantage of the pH- and chymotrypsin-sensitivities of extracellular pHluorin.

4. Antibody labeling assay for cell surface expression

Internalization of G-protein coupled receptors after being activated is one of the important mechanisms of receptor desensitization and subsequent down regulation. It is conceivable that, because of its high constitutive activity, GPR6 is continuously internalized and recycled to the surface. A small portion of GPR6 that is present on the cell surface may not be detected under the experimental conditions of the above three assays. Thus, we used an antibody labeling assay to determine if a small fraction of GPR6 is expressed at the cell surface and is being recycled. A high affinity GFP antibody is likely to bind all GPR6 receptors that are at the cell surface at any point during the period of antibody incubation. Furthermore the antibody can be labeled with fluorophores that have significantly greater quantum yield (brightness) than pHluorin, increasing the sensitivity of detection.

All other protocol details of the above you might find in the original manuscript entitled: “Methods to detect cell surface expression and constitutive activity of GPR6” by B. Prasad et al, published in Methods Enzymol. 2010; 484: 179–195. doi: 10.1016/B978-0-12-381298-8.00010-1

PMCID: PMC3932180

NIHMSID: NIHMS337888

Best wishes,

Ilya

Thanks a lot sir

OK