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.
Highlights
G-protein coupled receptors are involved in many key biological processes such as smell, taste and vision
All proteins stained by silver nitrate were revealed with anti-c-myc antibodies indicating that the three G-protein coupled receptors (GPCRs) preparations used to immunize mice were virtually pure
Silver nitrate dyeing as well as anti-c-myc staining, revealed bands with apparent molecular weights of 47 kDa, 40 kDa and 47.5 kDa corresponding to the calculated size of human neuropeptide FF receptor type 2 (hNPFFR2) (Fig. 1a), human k opioid receptor (hKOR) (Fig. 1b) and human m opioid receptor (hMOR) (Fig. 1c) respectively (Table 1)
Summary
G-protein coupled receptors are involved in many key biological processes such as smell, taste and vision. The remaining 40% are classified in five main families under the GRAFS system (Glutamate, Rhodopsin, Adhesion, Frizzled/taste and Secretin) [1,2] In line with their pivotal role in a number of physiological processes, GPCRs have been found dysregulated in several human pathologies including cardiovascular and gastrointestinal diseases, nervous and immune disorders and cancers. As recently reported for a number of GPCRs including opioid receptors [16], commercial available polyclonal antibodies often display non-specific reactivities and/or cross-reactivities with other plasma membrane proteins making it difficult to clearly distinguish a specific antibody-receptor binding. A recent study, comparing the specificity of a number of commercial anti-opioid receptor antibodies, has shown that all the antibodies revealed numerous non-specific bands including a band at the expected molecular weight in both wild-type CHO cells (negative control) and GPCR-expressing CHO cells as assessed by western-blotting [23]. This method successfully applied to the human neuropeptide FF receptor type 2 (hNPFFR2), the human k opioid receptor (hKOR) and the human m opioid receptor (hMOR) might be extended to a wide range of other GPCRs and applicable in most laboratories
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