A new approach to analyze cell surface protein complexes reveals specific heterodimeric metabotropic glutamate receptors.

G-protein-coupled receptors (GPCRs) can form heteromeric complexes. Herein, we describe a new approach to test the heteromerization of 2 receptors, or 2 receptor subunits, and to study the stoichiometry of the resulting complexes. As a proof-of-concept study, we investigated whether metabotropic glutamate receptors (mGluRs), in addition to being well-known homodimers, can form heteromers. To that aim, we combine the benefits of time-resolved fluorescence resonance energy transfer (trFRET) with the specific, cell-surface labeling of SNAP- and CLIP-tagged rat mGluR subunits, expressed in a mammalian cell line. First, we show that mGlu2 and mGlu4 subunits (but not mGlu2 and mGlu1) can heteromerize. Moreover, our trFRET data are consistent with mGluR subunits forming strict homodimeric receptors on single expression, and a combination of strict heterodimeric and strict homodimeric receptors on coexpression. Second, a comprehensive analysis reveals that from the 21 possible pairs of 2 mGluR subunits out of 7 subtypes (mGlu1 to 8, but not 6), only 11 are able to form heterodimers. These findings were further validated by biochemical and functional complementation studies. In addition to describing a new method to analyze cell-surface receptor complexes, our data reveal a new level of complexity within the mGluR family.