In situ visualization of opioid and cannabinoid drug effects using phosphosite-specific GPCR antibodies

Abstract G protein-coupled receptors (GPCRs) are vital signal transducers that upon activation become phosphorylated on intracellular serine and threonine residues. Although antibodies that specifically recognize the phosphorylation state of GPCRs have been available for many years, efficient immunolocalization of phosphorylated receptors in their tissues of origin has remained elusive. Here we show that GPCR phosphorylation is very unstable during routine immunohistochemical procedures, necessitating the presence of appropriate phosphatase inhibitors throughout both fixation and staining procedures. We provide proof of concept using three out of four phosphorylation state-specific µ-opioid receptor antibodies and show that this approach can be readily extended to other prototypical GPCRs such as the CB1 cannabinoid receptor. In summary, this improved protocol will facilitate the widespread application of phosphorylation state-specific antibodies to monitor the physiological and pharmacological activation of endogenous GPCRs.