Article

The use of receptor-specific antibodies to study G-protein-coupled receptors.

Department of Pharmacology and Biological Chemistry, Box 1603, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.
Mount Sinai Journal of Medicine A Journal of Translational and Personalized Medicine (Impact Factor: 1.56). 08/2006; 73(4):673-81.
Source: PubMed

ABSTRACT The identification of G-protein-coupled receptor (GPCR) cDNAs has facilitated a number of studies characterizing the biochemical properties of the receptor protein. Most of these studies have used antibodies directed against the epitope-tagged receptor expressed in heterologous cells, because of the lack of sensitive and selective antibodies capable of recognizing endogenous receptors in their native state. In order to facilitate studies with endogenous receptors, efforts have been made to generate receptor-type selective, sensitive antibodies that are able to recognize endogenous receptors. In this review, we discuss the strategies as well as the details of the techniques used for the generation of monoclonal and polyclonal antibodies with a focus on family A GPCRs.

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    • "In this context, highly specific anti-GPCR antibodies may be particularly helpful to better define anatomical localization as well as biochemical and biological properties of the receptors targeted for therapy [3]. Antibodies may be used to reveal GPCR expression on living cells (as assessed by cytofluorometry or confocal microscopy) or on membrane extracts (Western blotting) as well as in situ on fixed tissue sections (immunochemistry). "
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    ABSTRACT: G-protein coupled receptors (GPCRs) play a major role in a number of physiological and pathological processes. Thus, GPCRs have become the most frequent targets for development of new therapeutic drugs. In this context, the availability of highly specific antibodies may be decisive to obtain reliable findings on localization, function and medical relevance of GPCRs. However, the rapid and easy generation of highly selective anti-GPCR antibodies is still a challenge. Herein, we report that highly specific antibodies suitable for detection of GPCRs in native and unfolded forms can be elicited by immunizing animals against purified full length denatured recombinant GPCRs. Contrasting with the currently admitted postulate, our study shows that an active and well-folded GPCR is not required for the production of specific anti-GPCR antibodies. This new immunizing strategy validated with three different human GPCR (μ-opioid, κ-opioid, neuropeptide FF2 receptors) might be generalized to other members of the GPCR family.
    PLoS ONE 09/2012; 7(9):e46348. DOI:10.1371/journal.pone.0046348 · 3.23 Impact Factor
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    • "ELISA to quantify CB1R expression in either U2OS cells co-expressing ProLink/Enzyme Donor (PK)-tagged human DOR, Enzyme Activator (EA)-tagged β-arrestin fusion protein and myc-tagged mouse CB1R and to quantify DOR expression in wild-type, CB1R−/− or DOR−/− cortical membranes was carried out as described [25], [33], [35] using rabbit anti-myc (1∶1000), rat anti-DOR (1∶500), and HRP-conjugated anti-rabbit (1∶2000) or anti-rat antibodies (1∶1000). ELISA to quantify, total CB1R levels in N2ACB1R, N2ACB1RDOR or N2ACB1RECE2 cells was carried out in cells permeabilized with ice-cold methanol for 5 min while cell surface CB1R levels were determined in non-permeabilized cells using anti-CB1R (1∶500) and HRP-conjugated anti-rabbit (1∶1000) antibodies. "
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    • "Due to the inherent problems in the production and purification of GPCRs, the use of the intact GPCR-proteins as antigen has been difficult. Hence, most antibodies recognizing GPCRs have so far been generated using synthetic peptide fragments of the receptor protein as antigens (Gupta and Devi, 2006; Mackrill, 2004). Zhang et al. (2004) further developed this strategy by synthetically producing cyclic peptides, thought to mimic the extracellular loops of the CCR5 receptor, for the selection of single-chain Fv (scFv) fragments. "
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