G protein coupled receptors (GPCRs) are the most frequently targeted protein family in clinical therapeutics. The molecular interactions between GPCRs can have a profound impact on their function and on the effects of drugs that target them. We examined the functional and pharmacological effects of interactions between two glutamate-activated GPCRs called mGluR1 and mGluR5, which are known to form homodimeric complexes (mGluR1-mGluR1 and mGluR5-mGluR5 dimers), are involved in many patho-physiological processes, and have widespread distribution in the central nervous system. Our data suggest that these receptors can interact functionally, and that this interaction alters their sensitivity to selective pharmacological agents that target them. We suggest a novel hypothesis to explain this interaction in which mGluR1 and mGluR5 dimers combine to form tetrameric (or possibly larger) complexes with the ability to trans-activate, such that ligand binding to one receptor may activate G protein through the other. Further, these putative tetrameric receptors seem to respond to some pharmacological agents differently than their homodimeric constituents. To address this hypothesis, we will pursue the following Specific Aims: 1) To characterize the unique pharmacological properties of mGluR1/5 heteromers using the rat superior cervical ganglion (SCG) neuronal expression system, 2) To confirm the pharmacological and signaling properties of mGluR1/5 heteromers by expression in HeLa cells and using a different effectors to assay receptor signaling, 3) To directly assess the ability of fluorescently-tagged mGluR1, mGluR5 and both receptors together to form dimeric, tetrameric, or higher order multimeric receptors using multi-photon Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS), and 4) To verify the physiological relevance of functional mGluR interactions in neurons that natively express both mGluR1 and mGluR5. PUBLIC HEALTH RELEVANCE: The proposed experiments will lead to a more thorough understanding of complex molecular interactions necessary for function of metabotropic glutamate receptors (mGluRs), a widespread family of receptors in the central nervous system. Because mGluRs are involved in many important physiological and pathological processes and are increasingly valued as therapeutic targets, these studies will impact public health issues that affect millions of Americans.