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.
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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 · 3.53 Impact Factor
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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 · 3.53 Impact Factor
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ABSTRACT: This paper reports some observations on the behaviour of a binary particle bed consisting of a mixture of small and large particles in the axial plane of a rotating drum. The effects of Froude number, particle diameter, filling ratio and initial particle concentration on axial segregation are evaluated using polymer powders. The results show that the number of segregation bands formed depends strongly on bed composition and processing conditions. Based on the experimental results, a segregation model to quantify axial dispersion is proposed.Graphical AbstractThis paper reports on mixing a binary particle bed of small and large polymer powders in a rotating drum. The effects of Froude number, particle diameter, filling ratio and initial particle concentration on segregation and mixing degree are presented and a dynamic model is proposed to determine the optimum mixing time.Research Highlights► Video imaging is used to study the mixing of polymer powders in a rotating cylinder. ► The effect of processing conditions on segregation is presented. ► A second order model is proposed to determine the mixing quality with time. ► The optimum mixing time to get the best homogeneous mixture is determined.Powder Technology 02/2011; 207(1-3):324-334. DOI:10.1016/j.powtec.2010.11.014 · 2.27 Impact Factor