The use of receptor-specific antibodies to study G-protein-coupled receptors.
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.
- SourceAvailable from: Sophia Hober
[Show abstract] [Hide abstract]
- "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. "
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 · 2.01 Impact Factor
[Show abstract] [Hide abstract]
- "If these would be reliable, they would be highly important tools for pharmacological studies. Technical approaches for the generation and validation of receptor antibodies have been reviewed comprehensively (Gupta and Devi 2006; Mackrill 2004). Doubts about the usefulness of antibodies against GPCR (O'Connell et al. 2006; Pradidarcheep et al. 2008; Rhodes and Trimmer 2006), receptors from the ligand-gated ion channel family (Herber et al. 2004) or transmitter transporters (Chen et al. 2004) have been raised in the past and have even led to the retraction of papers (Hawes and Picciotto 2005). "
ABSTRACT: A cluster of manuscripts in this issue of the Journal highlights a lack of selectivity of 49 antibodies against 19 subtypes of alpha(1)- and beta-adrenoceptors, muscarinic, dopamine and galanin receptors as well as vanilloid (TRPV1) receptors. Taken together these data demonstrate that lack of selectivity appears to be the rule rather than the exception for antibodies against G-protein-coupled and perhaps also other receptors. Thus, the previously often applied validation of such antibodies by the disappearance of staining in the presence of blocking peptide, i.e. the antigen against which the antibody was raised, alone is insufficient to demonstrate specificity. We propose that receptor antibodies should be validated by at least one of the following techniques: a) disappearance of staining in knock-out animals of the target receptor, b) reduction of staining upon knock-down approaches such as siRNA treatment, c) selectivity of staining in immunoblots or immunocytochemistry for the target receptor vs. related subtypes when expressed in the same cell line and/or d) antibodies raised against multiple distinct epitopes of a receptor yielding very similar staining patterns. Other issues of consideration to obtain reliable results based on receptor antibodies in applications such as immunohistochemistry or immunoblotting are also being discussed.Archiv für Experimentelle Pathologie und Pharmakologie 02/2009; 379(4):385-8. DOI:10.1007/s00210-009-0395-y · 2.36 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The function and distribution of alpha1-adrenergic receptor (AR) subtypes in prostate cancer cells is well characterized. Previous studies have used RNA localization or low-avidity antibodies in tissue or cell lines to determine the alpha1-AR subtype and suggested that the alpha1A-AR is dominant. Two androgen-insensitive, human metastatic cancer cell lines DU145 and PC3 were used as well as the mouse TRAMP C1-C3 primary and clonal cell lines. The density of alpha1-ARs was determined by saturation binding and the distribution of the different alpha1-AR subtypes was examined by competition-binding experiments. In contrast to previous studies, the major alpha1-AR subtype in DU145, PC3 and all of the TRAMP cell lines is the alpha1B-AR. DU145 cells contained 100% of the alpha1B-AR subtype, whereas PC3 cells were composed of 21% alpha1 A-AR and 79% alpha1B-AR. TRAMP cell lines contained between 66% and 79% of the alpha1B-AR with minor fractions of the other two subtypes. Faster doubling time in the TRAMP cell lines correlated with decreasing alpha 1B-AR and increasing alpha1 A- and alpha1D-AR densities. Transfection with EGFP-tagged alpha1B-ARs revealed that localization was mainly intracellular, but the majority of the receptors translocated to the cell surface after extended preincubation (18 hr) with either agonist or antagonist. Localization was confirmed by ligand-binding studies and inositol phosphate assays where prolonged preincubation with either agonist and/or antagonist increased the density and function of alpha 1-ARs, suggesting that the native receptors were mostly intracellular and nonfunctional. Our studies indicate that alpha1B-ARs are the major alpha1-AR subtype expressed in DU145, PC3, and all TRAMP cell lines, but most of the receptor is localized in intracellular compartments in a nonfunctional state, which can be rescued upon prolonged incubation with any ligand.Journal of Receptor and Signal Transduction Research 02/2007; 27(1):27-45. DOI:10.1080/10799890601087487 · 1.61 Impact Factor