Jonathan A. Javitch

Functional Selectivity and Classical Concepts of Quantitative Pharmacology

The concept of intrinsic efficacy has been enshrined in pharmacology for half of a century, yet recent data have revealed that many ligands can differentially activate signaling pathways mediated via a single G protein-coupled receptor in a manner that challenges the traditional definition of intrinsic efficacy. Some terms for this phenomenon include functional selectivity, agonist-directed trafficking, and biased agonism. At the extreme, functionally selective ligands may be both agonists and antagonists at different functions mediated by the same receptor. Data illustrating this phenomenon are presented from serotonin, opioid, dopamine, vasopressin, and adrenergic receptor systems. A variety of mechanisms may influence this apparently ubiquitous phenomenon. It may be initiated by differences in ligand-induced intermediate conformational states, as shown for the β2-adrenergic receptor. Subsequent mechanisms that may play a role include diversity of G proteins, scaffolding and signaling partners, and receptor oligomers. Clearly, expanded research is needed to elucidate the proximal (e.g., how functionally selective ligands cause conformational changes that initiate differential signaling), intermediate (mechanisms that translate conformation changes into differential signaling), and distal mechanisms (differential effects on target tissue or organism). Besides the heuristically interesting nature of functional selectivity, there is a clear impact on drug discovery, because this mechanism raises the possibility of selecting or designing novel ligands that differentially activate only a subset of functions of a single receptor, thereby optimizing therapeutic action. It also may be timely to revise classic concepts in quantitative pharmacology and relevant pharmacological conventions to incorporate these new concepts.

Structure of the human dopamine d3 receptor in complex with a d2/d3 selective antagonist.

Tweaking Dopamine Reception Dopamine modulates many cognitive and emotional functions of the human brain by activating G protein–coupled receptors. Antipsychotic drugs that block two of the receptor subtypes are used to treat schizophrenia but have multiple side effects. Chien et al. (p. 1091; see the Research Article by Wu et al.) resolved the crystal structure of one receptor in complex with a small-molecule inhibitor at 3.15 angstrom resolution. Homology modeling with other receptor subtypes might be a promising route to reveal potential structural differences that can be exploited in the design of selective therapeutic inhibitors having fewer side effects. Discovery of a binding site in the extracellular domain of a dopamine receptor offers hope for more selective therapeutics. Dopamine modulates movement, cognition, and emotion through activation of dopamine G protein–coupled receptors in the brain. The crystal structure of the human dopamine D3 receptor (D3R) in complex with the small molecule D2R/D3R-specific antagonist eticlopride reveals important features of the ligand binding pocket and extracellular loops. On the intracellular side of the receptor, a locked conformation of the ionic lock and two distinctly different conformations of intracellular loop 2 are observed. Docking of R-22, a D3R-selective antagonist, reveals an extracellular extension of the eticlopride binding site that comprises a second binding pocket for the aryl amide of R-22, which differs between the highly homologous D2R and D3R. This difference provides direction to the design of D3R-selective agents for treating drug abuse and other neuropsychiatric indications.

β2 Adrenergic Receptor Activation MODULATION OF THE PROLINE KINK IN TRANSMEMBRANE 6 BY A ROTAMER TOGGLE SWITCH

In many rhodopsin-like G-protein-coupled receptors, agonist binding to a cluster of aromatic residues in TM6 may promote receptor activation by altering the configuration of the TM6 Pro-kink and by the subsequent movement of the cytoplasmic end of TM6 away from TM3. We hypothesized that the highly conserved Cys6.47, in the vicinity of the conserved Pro6.50, modulates the configuration of the aromatic cluster and the TM6 Pro-kink through specific interactions in its different rotamer configurations. In the β2 adrenergic receptor, mutation of Cys6.47 to Thr, which in an α-helix has a different rotamer distribution from Cys and Ser, produced a constitutively active receptor, whereas the Ser mutant was similar to wild-type receptor. Use of the biased Monte Carlo technique of Conformational Memories showed that the rotamer changes among Cys/Ser/Thr6.47, Trp6.48, and Phe6.52 are highly correlated, representing a rotamer “toggle switch” that may modulate the TM6 Pro-kink. Differential modulation of the accessibility of Cys6.47 and an engineered Cys6.52 in wild type and a constitutively active background provides experimental support for the association of this rotamer switch with receptor activation.

Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue

Neurotransmitter/Na+ symporters (NSSs) terminate neuronal signalling by recapturing neurotransmitter released into the synapse in a co-transport (symport) mechanism driven by the Na+ electrochemical gradient. NSSs for dopamine, noradrenaline and serotonin are targeted by the psychostimulants cocaine and amphetamine, as well as by antidepressants. The crystal structure of LeuT, a prokaryotic NSS homologue, revealed an occluded conformation in which a leucine (Leu) and two Na+ are bound deep within the protein. This structure has been the basis for extensive structural and computational exploration of the functional mechanisms of proteins with a LeuT-like fold. Subsequently, an ‘outward-open’ conformation was determined in the presence of the inhibitor tryptophan, and the Na+-dependent formation of a dynamic outward-facing intermediate was identified using electron paramagnetic resonance spectroscopy. In addition, single-molecule fluorescence resonance energy transfer imaging has been used to reveal reversible transitions to an inward-open LeuT conformation, which involve the movement of transmembrane helix TM1a away from the transmembrane helical bundle. We investigated how substrate binding is coupled to structural transitions in LeuT during Na+-coupled transport. Here we report a process whereby substrate binding from the extracellular side of LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein. In the presence of alanine, a substrate that is transported ∼10-fold faster than leucine, we observed alanine-induced dynamics in the intracellular gate region of LeuT that directly correlate with transport efficiency. Collectively, our data reveal functionally relevant and previously hidden aspects of the NSS transport mechanism that emphasize the functional importance of a second substrate (S2) binding site within the extracellular vestibule. Substrate binding in this S2 site appears to act cooperatively with the primary substrate (S1) binding site to control intracellular gating more than 30 Å away, in a manner that allows the Na+ gradient to power the transport mechanism.

Allosteric communication between protomers of dopamine class A GPCR dimers modulates activation.

A major obstacle to understanding the functional importance of dimerization between Class A G protein-coupled receptors (GPCRs) has been the methodological limitation in achieving control of the identity of the components comprising the signaling unit. We have developed a functional complementation assay that enables such control and illustrate it for the human dopamine D2 receptor. The minimal signaling unit, two receptors and a single G protein, is maximally activated by agonist binding to a single protomer, which suggests an asymmetrical activated dimer. Inverse agonist binding to the second protomer enhances signaling, whereas agonist binding to the second protomer blunts signaling. Ligand-independent constitutive activation of the second protomer also inhibits signaling. Thus, GPCR dimer function can be modulated by the activity state of the second protomer, which for a heterodimer may be altered in pathological states. Our novel methodology also makes possible the characterization of signaling from a defined heterodimer unit.

Dopamine D2 receptors form higher order oligomers at physiological expression levels

G‐protein‐coupled receptors are generally thought to be organized as dimers; whether they form higher order oligomers is a topic of much controversy. We combined bioluminescence/fluorescence complementation and energy transfer to demonstrate that at least four dopamine D2 receptors are located in close molecular proximity in living mammalian cells, consistent with their organization as higher order oligomers at the plasma membrane. This implies the existence of multiple receptor interfaces. In addition to the symmetrical interface in the fourth transmembrane segment (TM4) we identified previously by cysteine (Cys) crosslinking, we now show that a patch of residues at the extracellular end of TM1 forms a second symmetrical interface. Crosslinking of D2 receptor with Cys substituted simultaneously into both TM1 and TM4 led to higher order species, consistent with our novel biophysical results. Remarkably, the rate and extent of crosslinking at both interfaces were unaltered over a 100‐fold range of receptor expression. Thus, at physiological levels of expression, the receptor is organized in the plasma membrane into a higher order oligomeric structure.

Building a new conceptual framework for receptor heteromers

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.

The Fourth Transmembrane Segment Forms the Interface of the Dopamine D2 Receptor Homodimer

Considerable evidence suggests that G-protein-coupled receptors form homomeric and heteromeric dimersin vivo. Unraveling the structural mechanism for cross-talk between receptors in a dimeric complex must start with the identification of the presently unknown dimer interface. Here, by using cysteine cross-linking, we identify the fourth transmembrane segment (TM4) as a symmetrical dimer interface in the dopamine D2 receptor. Cross-linking is unaffected by ligand binding, and ligand binding and receptor activation are unaffected by cross-linking, suggesting that the receptor is a constitutive dimer. The accessibility of adjacent residues in TM4, however, is affected by ligand binding, implying that the interface has functional significance.

Time-resolved FRET between GPCR ligands reveals oligomers in native tissues

G protein-coupled receptor (GPCR) oligomers have been proposed to play critical roles in cell signaling, but confirmation of their existence in a native context remains elusive, as no direct interactions between receptors have been reported. To demonstrate their presence in native tissues, we developed a time-resolved FRET strategy that is based on receptor labeling with selective fluorescent ligands. Specific FRET signals were observed with four different receptors expressed in cell lines, consistent with their dimeric or oligomeric nature in these transfected cells. More notably, the comparison between FRET signals measured with sets of fluorescent agonists and antagonists was consistent with an asymmetric relationship of the two protomers in an activated GPCR dimer. Finally, we applied the strategy to native tissues and succeeded in demonstrating the presence of oxytocin receptor dimers and/or oligomers in mammary gland.

The binding sites for cocaine and dopamine in the dopamine transporter overlap

Cocaine is a widely abused substance with psychostimulant effects that are attributed to inhibition of the dopamine transporter (DAT). We present molecular models for DAT binding of cocaine and cocaine analogs constructed from the high-resolution structure of the bacterial transporter homolog LeuT. Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine, as well as for benztropine-like DAT inhibitors. We validated our models by detailed mutagenesis and by trapping the radiolabeled cocaine analog [3H]CFT in the transporter, either by cross-linking engineered cysteines or with an engineered Zn2+-binding site that was situated extracellularly to the predicted common binding pocket. Our data demonstrate the molecular basis for the competitive inhibition of dopamine transport by cocaine.