The use of receptor-specific antibodies to study G-protein-coupled receptors
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.
Available from: Franck Talmont
- "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 . 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
Available from: Avi Ma'ayan
- "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 , ,  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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ABSTRACT: A fundamental question in G protein coupled receptor biology is how a single ligand acting at a specific receptor is able to induce a range of signaling that results in a variety of physiological responses. We focused on Type 1 cannabinoid receptor (CB₁R) as a model GPCR involved in a variety of processes spanning from analgesia and euphoria to neuronal development, survival and differentiation. We examined receptor dimerization as a possible mechanism underlying expanded signaling responses by a single ligand and focused on interactions between CB₁R and delta opioid receptor (DOR). Using co-immunoprecipitation assays as well as analysis of changes in receptor subcellular localization upon co-expression, we show that CB₁R and DOR form receptor heteromers. We find that heteromerization affects receptor signaling since the potency of the CB₁R ligand to stimulate G-protein activity is increased in the absence of DOR, suggesting that the decrease in CB₁R activity in the presence of DOR could, at least in part, be due to heteromerization. We also find that the decrease in activity is associated with enhanced PLC-dependent recruitment of arrestin3 to the CB₁R-DOR complex, suggesting that interaction with DOR enhances arrestin-mediated CB₁R desensitization. Additionally, presence of DOR facilitates signaling via a new CB₁R-mediated anti-apoptotic pathway leading to enhanced neuronal survival. Taken together, these results support a role for CB₁R-DOR heteromerization in diversification of endocannabinoid signaling and highlight the importance of heteromer-directed signal trafficking in enhancing the repertoire of GPCR signaling.
PLoS ONE 01/2012; 7(1):e29239. DOI:10.1371/journal.pone.0029239 · 3.23 Impact Factor
Available from: Sophia Hober
- "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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ABSTRACT: Antibodies are important tools for the study of G-protein-coupled receptors, key proteins in cellular signaling. Due to their large hydrophobic membrane spanning regions and often very short loops exposed on the surface of the cells, generation of antibodies able to recognize the receptors in the endogenous environment has been difficult. Here, we describe an antigen-design method where the extracellular loops and N-terminus are combined to a single antigen for generation of antibodies specific to three selected GPCRs: NPY5R, B2ARN and GLP1R. The design strategy enabled straightforward antigen production and antibody generation. Binding of the antibodies to intact receptors was analyzed using flow cytometry and immunofluorescence based confocal microscopy on A-431 cells overexpressing the respective GPCR. The antibody-antigen interactions were characterized using epitope mapping, and the antibodies were applied in immunohistochemical staining of human tissues. Most of the antibodies showed specific binding to their respective overexpressing cell line but not to the non-transfected cells, thus indicating binding to their respective target receptor. The epitope mapping showed that sub-populations within the purified antibody pool recognized different regions of the antigen. Hence, the genetic combination of several different epitopes enables efficient generation of specific antibodies with potential use in several applications for the study of endogenous receptors.
Journal of immunological methods 05/2011; 370(1-2):14-23. DOI:10.1016/j.jim.2011.05.001 · 1.82 Impact Factor
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