Abstract
Over the last decade an increasing number of studies have focused on the ability of G protein-coupled receptors to form heteromers and explored how receptor heteromerization modulates the binding, signaling and trafficking properties of individual receptors. Most of these studies were carried out in heterologous cells expressing epitope tagged receptors. Very little information is available about the in vivo physiological role of G protein-coupled receptor heteromers due to a lack of tools to detect their presence in endogenous tissue. Recent advances such as the generation of mouse models expressing fluorescently labeled receptors, of TAT based peptides that can disrupt a given heteromer pair, or of heteromer-selective antibodies that recognize the heteromer in endogenous tissue have begun to elucidate the physiological and pathological roles of receptor heteromers. In this review we have focused on heteromer-selective antibodies and describe how a subtractive immunization strategy can be successfully used to generate antibodies that selectively recognize a desired heteromer pair. We also describe the uses of these antibodies to detect the presence of heteromers, to study their properties in endogenous tissues, and to monitor changes in heteromer levels under pathological conditions. Together, these findings suggest that G protein-coupled receptor heteromers represent unique targets for the development of drugs with reduced side-effects.
Highlights
Since the first report showing that metabotropic GABAB receptors, members of the family C of G protein-coupled receptors (GPCRs), form constitutive heteromers (White et al, 1998; Kuner et al, 1999; Pin et al, 2009) an increasing number of studies have provided evidence suggesting that other GPCRs, those belonging to family A, heteromerize (Albizu et al, 2010; Gomes et al, 2013a; Hiller et al, 2013; Szafran et al, 2013)
In this review we describe how a subtractive immunization strategy can be successfully used to generate monoclonal antibodies that are selective for a given heteromer pair and that can be useful for examination of endogenous heteromers
Even though the procedure is time consuming, and requires a number of controls during screening procedures for determining the heteromer-selectivity of the antibodies, there are many advantages to developing the heteromer selective antibodies. These include the fact that they recognize a unique epitope that is present only in cells/tissues expressing the heteromer of interest, and they could be used to map the targeted heteromer in endogenous tissue and to monitor changes in heteromer levels during pathological conditions
Summary
Since the first report showing that metabotropic GABAB receptors, members of the family C of G protein-coupled receptors (GPCRs), form constitutive heteromers (White et al, 1998; Kuner et al, 1999; Pin et al, 2009) an increasing number of studies have provided evidence suggesting that other GPCRs, those belonging to family A, heteromerize (Albizu et al, 2010; Gomes et al, 2013a; Hiller et al, 2013; Szafran et al, 2013). In order to improve our chances of generating heteromer-selective antibodies we used a subtractive immunization strategy (Salata et al, 1992; Sleister and Rao, 2001, 2002) that involves two major steps: (i) tolerization of mice to unwanted epitopes, and (ii) immunization with membranes expressing the heteromer pair of interest. One needs to be careful about selecting the screening technique to allow for detection of the antigen under the assay of choice Using this subtractive immunization strategy we successfully generated antibodies selective for either μOR-δOR, δOR-κOR, δOR-CB1R, or AT1R-CB1R heteromers (Gupta et al, 2010; Rozenfeld et al, 2011; Berg et al, 2012; Bushlin et al, 2012). A number of immunohistochemical studies showed that both
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