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Determining the oligomer number of native GPCR using florescence correlation spectroscopy and drug-induced inactivation-reactivation.

GPCRs are a major family of homologous proteins and are key mediators of the effects of numerous endogenous neurotransmitters, hormones, cytokines, therapeutic drugs, and drugs-of-abuse. Despite the enormous amount of research on the pharmacological and biochemical properties of GPCRs, there is surprisingly little information on GPCR dimer structure and function in primary cell culture or in vivo. We have used two novel approaches to develop methods to detect and study GPCR dimer function: FCS/PCH and "inactivation-reactivation". This review will focus on the data we have developed and our interpretations of those data. Using FCS/PCH 5-HT2C receptors have been detected directly and appear to exist as dimers, consistent with the inactivation-reactivation data on 5-HT7 and 5-HT2A receptors. Studies of the 5-HT7 and 5-HT2A serotonin receptors have revealed that binding of a pseudo-irreversible antagonist ("inactivator") to one of the orthosteric sites of a homodimer abolishes all receptor activity, and subsequent binding of a competitive antagonist to the orthosteric site of the second protomer releases the inactivator, allowing the receptor to return to an active state. This approach demonstrates allosteric crosstalk between protomers of native GPCR homodimers, indicating that GPCRs do exist and function as homodimers in both recombinant cells and rat primary astrocytes. This technique can be applied universally using intact recombinant or primary cells in culture, membrane homogenate preparations and, potentially, in vivo. This approach can be applied to heterodimers as well as homodimers and may aid in the development of novel drugs with heterodimer selectivity.