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Technologies that bias GPCR expression for formation of heterodimers show that, when heterodimerized, α_2C-AR and AT-1R exhibit atypical G_s-cAMP-PKA signaling upon ligand stimulation compared to either parent receptor expressed alone and mimic activation associated with arterial hypertension.

The dopamine transporter (DAT), a regulator of dopamine homeostasis in the brain, and sigma-1 receptor (σ₁R), an endoplasmic reticulum membrane protein, are both implicated in drug addiction. In this work, the authors investigate how σ₁R modulates DAT response to methamphetamine.

Metabotropic glutamate (mGlu) receptors form both homo- and heterodimers that respond to ligands differently. Here, the authors elucidate the mechanism of activation and differential pharmacology in mGlu_2/4 heteromers, which may guide targeted therapy.

Structures of the human calcium-sensing receptor can be bound into complex with G proteins from three different Gα subtypes while maintaining G-protein-binding specificity.

Amphetamines are known to enhance extracellular dopamine levels, but the underlying mechanisms are unclear. Utilising a new pH biosensor for synaptic vesicles, the authors show that amphetamines diminish vesicle pH gradients, disrupting dopamine packaging and leading to increased neurotransmitter release.

A suite of generally applicable methods and tools, developed to enable single-molecule FRET-based studies of transmembrane proteins diffusing in the cell membrane of living cells, was used to study the oligomerization and dynamics of GPCRs.

Ibogaine is a natural substance that interrupts opioid addiction but has cardiac risks. This article introduces novel ibogaine analogs that show reduced cardiac risk and enhanced neuroplasticity and therapeutic-like effects in models of opioid use disorder.

The structure of the GABA_B receptor in an inactive state reveals, amongst other features, a latch between the two subunits that locks the transmembrane domain interface, and the presence of large phospholipids that may modulate receptor function.

Conjugation of triplet-state quenchers to the small organic cyanine fluorophore, Cy5, increases photostability without affecting its spectral characteristics. This allows longer fluorescence imaging with a concomitant reduction in blinking both in vitro and in living cells.

Neurotransmitter:sodium symporters (NSS) use energy derived from sodium gradients to power transport of their respective neurotransmitters. But, whereas most eukaryotic NSS transporters are chloride-dependent, the prokaryotic homologue LeuT is not. Structural and mechanistic knowledge of LeuT, and a chloride transporter from an entirely different family, is applied to mammalian NSS transporters and finds that transported chloride ions serve to neutralise the positive charge carried by the sodium ions.

Neurotransmitter:sodium symporters (NSS) modulate the duration and magnitude of signaling via the sodium-coupled reuptake of neurotransmitters. Here the authors describe quantitative single molecule imaging of ligand-induced, functional dynamics of both intracellular and extracellular surfaces of LeuT, further defining the mechanism for NSS transport.

Mitragynine (MG) is an indole alkaloid from kratom plant that binds opioid receptors and as such presents a scaffold for the development of atypical opioid receptor modulators. Here, the authors report a synthetic method for selective functionalization of the C11 position of MG, and show that this position is essential for fine-tuning opioid receptor signaling efficacy.

Imaging of substrate transport by individual MhsT transporters, members of the neurotransmitter:sodium symporter family of secondary transporters, at single- and multi-turnover resolution reveals that the rate-limiting step varies with the identity of the transported substrate.

A recently identified pertussis toxin-like AB5 toxin, OZITX, is found to inhibit Gαi/o and Gαz G proteins. In combination with directed mutations, it is a useful tool for interrogating Gαi/o/z G protein subunits individually.

Mutations in CNTNAP2 have been associated with a syndromic form of Autism Spectrum Disorder. Here the authors show that forebrain organoids generated from induced pluripotent stem cells of patients with a syndromic form of ASD with a homozygous truncating mutation in CNTNAP2 displayed an increase in volume and total cell number, which is driven by abnormal cellular proliferation and neurogenesis.

Among the adhesion receptor class of GPCRs, which are understudied, the adhesion receptor ADGRL3 can be activated by its own tethered agonist and couples to G protein G12/13 and somewhat to Gq.

Neurotransmitter:sodium symporters (NSS) serve as targets for drugs including antidepressants and psychostimulants. Here authors report the X-ray structure of the prokaryotic NSS member, LeuT, in a Na⁺/substrate-bound, inward-facing occluded conformation which is a key intermediate in the LeuT transport cycle.

Here, the X-ray crystal structure of TrkH is solved, a protein that mediates potassium uptake in Vibrio parahaemolyticus, a bacterium. The selectivity filter of the ion transporter is very similar to what is seen in (much simpler) potassium ion channels, but it is significantly shorter. Biochemical studies were performed to explore the molecular basis of K⁺ selectivity, and it is believed that a novel gating mechanism is involved.

Neurotransmitter:Na⁺ symporters (NSS) remove neurotransmitters from the synapse in a reuptake process that is driven by the Na⁺ gradient. Here, single-molecule fluorescence imaging assays have been combined with molecular dynamics simulations to probe the conformational changes that are associated with substrate binding and transport by a prokaryotic NSS homologue, LeuT. The findings are interpreted in the context of an allosteric mechanism that couples ion and substrate binding to transport.

Based on a BRET readout, dopamine D2 receptor agonist NPA is more potent at activating Gα_i when the D2 receptor forms a heteromer with the related D1 receptor than if it forms D2 receptor homomers, suggesting that GPCR heteromerization can result in functional selectivity.

Biased agonists act at a receptor to preferentially induce distinct intracellular signalling responses over others. Here the authors show how kinetics of ligand binding and signaling responses greatly influence observed bias profiles, and hence must be considered when studying biased agonists.

D1-like dopamine receptors are coupled to Golf proteins in the dorsal striatum but Gs in cortical and other areas. Here, the authors demonstrate selective agonism of Gs-coupled versus Golf-coupled D1 receptors.

Na⁺and substrate symport through Tyt1, a prokaryotic neurotransmitter:sodium symporter, requires an inversely oriented H⁺ gradient, maintaining an ionic counterbalance during neurotransmitter translocation that is facilitated by negatively charged amino acid residues.

Evidence for the existence and importance of GPCR dimers and oligomers is mounting, but direct detection of these species has been challenging. The development of improved fluorescent ligands for time-resolved spectroscopy confirms their presence across GPCR families and in native tissue.

Crystal structures of the bacterial LeuT Na⁺-substrate symporter have revealed one substrate molecule in an occluded, centrally located binding site, whereas subsequent studies identified a putative second substrate binding site. Now additional binding analyses demonstrate that the second substrate binding site can be obscured during the preparation of detergent-solubilized LeuT for crystallography, explaining the apparent discrepancy in the reported stoichiometry.

Single-molecule FRET imaging provides insights into the allosteric link between the ligand-binding and G-protein nucleotide-binding pockets of the β₂ adrenergic receptor (β₂AR) and improved understanding of the G-protein activation mechanism.

A compound previously identified as a dopamine D2 receptor allosteric modulator was found to be a bitopic ligand that binds the orthosteric and allosteric sites to allow binding to one D2 protomer and allosteric modulation of the associated protomer.

PIP₂ binds via electrostatic interactions to the N terminus of the dopamine transporter (DAT) to modulate its function in dopamine efflux. This regulation is necessary for Drosophila locomotor activity induced by the DAT substrate amphetamine.

Crystal structures of MhsT, a bacterial member of the neurotransmitter/sodium symporter family, in an occluded, inward-facing state with bound sodium and substrate reveal conformational changes during the transport cycle that provide new insights into the mechanism of cytoplasmic sodium release.

LeuT is a bacterial sodium/leucine symporter, related to human neurotransmitters targeted by antidepressant drugs. Now spin labeling and EPR analysis on the dynamics in the extracellular vestibule of LeuT reveal the conformational changes caused by Na⁺ and Leu binding that drive the transport cycle.

Dopamine D2 receptor activity in the nucleus accumbens is associated with regulation of motivated responding. Here the authors show that overexpression of D2 receptors specifically in ventral striatal projection neurons leads to an increase in the willingness to work by reducing inhibitory transmission to ventral pallidal neurons.

This paper demonstrates, using structural and functional analysis of the GBR2 ectodomain, that this GABA_B receptor subunit potentiates ligand binding by selectively stabilizing the agonist-bound conformation of the GBR1 subunit.

Atypical antipsychotics show reduced extrapyramidal side effects compared to first generation drugs. Here the authors use time-resolved FRET to measure binding kinetics, and show that side effects correlate with drug association rates to the D₂ receptor, while dissociation rates correlate with prolactin elevation.

Beuming and colleagues determined that the binding site for cocaine overlaps with that of dopamine on the dopamine transporter. Detailed modeling and mutagenesis experiments revealed that this site is deeply buried amongst several transmembrane domains.

The authors report that the protein Flotillin-1/Reggie-2 (Flot1) is required for PKC-regulated internalization of the dopamine transporter (DAT) and the glial glutamate transporter EAAT2. Flot1 was required to localize DAT within plasma membrane microdomains in stable cell lines, and for amphetamine-induced reverse transport of dopamine in neurons.

LeuT is a Na⁺-coupled amino acid transporter that is similar in sequence and function to eukaryotic neurotransmitter/sodium symporters, which are active in reuptake of neurotransmitters from the synapse. Distance measurements between spin-label pairs are used to identify ligand-dependent structural transitions in LeuT.

Probes that reversibly recognize oligohistidine sequences engineered into proteins can provide insights into the structure and location of proteins in living cells.

Receptor heteromers constitute a new area of research that is reshaping our thinking about biochemistry, cell biology, pharmacology and drug discovery. In this commentary, we recommend clear definitions that should facilitate both information exchange and research on this growing class of transmembrane signal transduction units and their complex properties. We also consider research questions underlying the proposed nomenclature, with recommendations for receptor heteromer identification in native tissues and their use as targets for drug development.