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Recent studies have proposed that heteromers of μ-opioid receptors (MORs) and galanin Gal1 receptors (Gal1Rs) localized in the mesencephalon mediate the dopaminergic effects of opioids. The present study reports converging evidence, using a peptide-interfering approach combined with biophysical and biochemical techniques, including total internal reflection fluorescence microscopy, for a predominant homodimeric structure of MOR and Gal1R when expressed individually, and for their preference to form functional heterotetramers when co-expressed. Results show that a heteromerization-dependent change in the Gal1R homodimeric interface leads to a switch in G-protein coupling from inhibitory Gi to stimulatory Gs proteins. The MOR-Gal1R heterotetramer, which is thus bound to Gs via the Gal1R homodimer and Gi via the MOR homodimer, provides the framework for a canonical Gs-Gi antagonist interaction at the adenylyl cyclase level. These novel results shed light on the intense debate about the oligomeric quaternary structure of G protein-coupled receptors, their predilection for heteromer formation, and the resulting functional significance.

Gαs (Gs) and Gαolf (Golf) are highly homologous G-protein α subunits that activate adenylate cyclase, thereby serving as crucial mediators of intracellular signaling. Because of their dramatically different brain expression patterns, we studied similarities and differences between their activation processes with the aim of comparing their receptor coupling mechanisms. We engineered novel luciferase- and Venus-fused Gα constructs that can be used in bioluminescence resonance energy transfer assays. In conjunction with molecular simulations, these novel biosensors were used to determine receptor activation–induced changes in conformation. Relative movements in Gs were consistent with the crystal structure of β2 adrenergic receptor in complex with Gs. Conformational changes in Golf activation are shown to be similar to those in Gs. Overall the current study reveals general similarities between Gs and Golf activation at the molecular level and provides a novel set of tools to search for Gs- and Golf-specific receptor pharmacology. In view of the wide functional and pharmacological roles of Gs- and Golf-coupled dopamine D1 receptor and adenosine A2A receptor in the brain and other organs, elucidating their differential structure–function relationships with Gs and Golf might provide new approaches for the treatment of a variety of neuropsychiatric disorders. In particular, these novel biosensors can be used to reveal potentially therapeutic dopamine D1 receptor and adenosine A2A receptor ligands with functionally selective properties between Gs and Golf signaling.

The excitotoxic cell death and the release of γ-amino-butyric acid (GABA) evoked by excitatory amino acids (EAAs) were comparatively examined in rat cortical sister cultures grown in serum-free (N2) and serum-supplemented (SSM) media. Cell death was induced by 24 h exposure to 1 mM N- methyl- d -aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) or kainate. [ 3 H]GABA release was evoked by 5 min exposure of preloaded cultures to 0.5 mM NMDA, AMPA or kainate. EAAs evoked remarkable GABA release in both N2 and SSM cultures, but caused toxic cell death in SSM cultures, only. Our findings indicate that functionally active EAA receptors do not necessarily mediate neurotoxicity and suggest that excitotoxicity can be prevented without blocking excitatory transmission.