Ulrik Gether

G Protein-coupled Receptors II. MECHANISM OF AGONIST ACTIVATION

The majority of transmembrane signal transduction in response to hormones and neurotransmitters is mediated by G proteincoupled receptors (GPCRs). Moreover, GPCRs are the principal signal transducers for the senses of sight and smell. GPCRs are characterized by seven membrane-spanning domains with an extracellular N terminus and a cytoplasmic C terminus (Fig. 1) (GPCR structure reviewed in Refs. 1–3). Based on certain key sequences, GPCRs can be divided into three major subfamilies, receptors related to rhodopsin (type A), receptors related to the calcitonin receptor (type B), and receptors related to the metabotropic receptors (type C). Of these, the rhodopsin subfamily is by far the largest and most extensively investigated and will be the focus of the present review. GPCR domains involved in ligand binding are nearly as diverse as the chemical structures of the known agonists (1, 3, 5). Small molecular weight ligands bind to sites within the hydrophobic core formed by the transmembrane (TM) a-helices, whereas binding sites for peptides and protein agonists include the N terminus and extracellular hydrophilic loops joining the transmembrane domains (2, 5). Domains critical for interaction with the G protein have been localized to the second and third cytoplasmic loops and the C terminus (2). High resolution structural analysis of GPCRs has been hindered by their low natural abundance and the difficulty in producing and purifying significant quantities of recombinant protein. A low resolution structure of rhodopsin obtained from electron diffraction of two-dimensional crystals has been useful in predicting the arrangement and relative orientation of the transmembrane domains (6, 7) (Fig. 2A). Mutagenesis studies aimed at identifying intramolecular interactions have provided evidence for a similar arrangement in other GPCRs (8–12), and several molecular models of different GPCRs have been developed (13–15). This review will examine current progress in addressing a fundamental question in the field of GPCR-mediated signal transduction: the nature of the physical changes in receptors that link agonist binding to G protein activation. It will explore the differences and similarities in the activation of rhodopsin and other GPCRs by focusing on two questions. What conformational changes take place during receptor activation? How do agonists induce these conformational changes in the receptor? Extended Ternary Complex Model Perhaps the most widely accepted model used to describe agonist activation of GPCRs is the ternary complex model, which accounts for the cooperative interactions among receptor, G protein, and agonist (16). This model has recently been extended to accommodate the observation that many receptors can activate G proteins in the absence of agonist and that mutations in different structural domains of the receptor can enhance this agonist-independent activity (17, 18). The extended ternary complex model also accounts for the effects of different classes of drugs (full agonists, partial agonists, neutral antagonists, and inverse agonists) on receptor signaling. The model proposes that the receptor exists in an equilibrium of two functionally distinct states: the inactive (R) and the active (R*) state. In the absence of agonists, the level of basal receptor activity is determined by the equilibrium between R and R*. The efficacy of ligands is thought to be a reflection of their ability to alter the equilibrium between these two states. Whereas most properties of GPCRs can be explained by this model, several studies suggest that a more complex model may be necessary (reviewed by Kenakin (19, 20)). Nevertheless, for the purpose of our discussion we will use the terms R to refer to the inactive conformation of the receptor and R* to refer to the conformation capable of activating G proteins.

Correlation between Genetic and Geographic Structure in Europe

Understanding the genetic structure of the European population is important, not only from a historical perspective, but also for the appropriate design and interpretation of genetic epidemiological studies. Previous population genetic analyses with autosomal markers in Europe either had a wide geographic but narrow genomic coverage [1, 2], or vice versa [3-6]. We therefore investigated Affymetrix GeneChip 500K genotype data from 2,514 individuals belonging to 23 different subpopulations, widely spread over Europe. Although we found only a low level of genetic differentiation between subpopulations, the existing differences were characterized by a strong continent-wide correlation between geographic and genetic distance. Furthermore, mean heterozygosity was larger, and mean linkage disequilibrium smaller, in southern as compared to northern Europe. Both parameters clearly showed a clinal distribution that provided evidence for a spatial continuity of genetic diversity in Europe. Our comprehensive genetic data are thus compatible with expectations based upon European population history, including the hypotheses of a south-north expansion and/or a larger effective population size in southern than in northern Europe. By including the widely used CEPH from Utah (CEU) samples into our analysis, we could show that these individuals represent northern and western Europeans reasonably well, thereby confirming their assumed regional ancestry.

SLC6 Neurotransmitter Transporters: Structure, Function, and Regulation

The neurotransmitter transporters (NTTs) belonging to the solute carrier 6 (SLC6) gene family (also referred to as the neurotransmitter-sodium-symporter family or Na+/Cl−-dependent transporters) comprise a group of nine sodium- and chloride-dependent plasma membrane transporters for the monoamine neurotransmitters serotonin (5-hydroxytryptamine), dopamine, and norepinephrine, and the amino acid neurotransmitters GABA and glycine. The SLC6 NTTs are widely expressed in the mammalian brain and play an essential role in regulating neurotransmitter signaling and homeostasis by mediating uptake of released neurotransmitters from the extracellular space into neurons and glial cells. The transporters are targets for a wide range of therapeutic drugs used in treatment of psychiatric diseases, including major depression, anxiety disorders, attention deficit hyperactivity disorder and epilepsy. Furthermore, psychostimulants such as cocaine and amphetamines have the SLC6 NTTs as primary targets. Beginning with the determination of a high-resolution structure of a prokaryotic homolog of the mammalian SLC6 transporters in 2005, the understanding of the molecular structure, function, and pharmacology of these proteins has advanced rapidly. Furthermore, intensive efforts have been directed toward understanding the molecular and cellular mechanisms involved in regulation of the activity of this important class of transporters, leading to new methodological developments and important insights. This review provides an update of these advances and their implications for the current understanding of the SLC6 NTTs.

Agonists induce conformational changes in transmembrane domains III and VI of the β2 adrenoceptor

Agonist binding to G protein‐coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that agonist binding to the β2 adrenoceptor induces a conformational change around 125Cys in transmembrane domain (TM) III and around 285Cys in TM VI. A series of mutant β2 adrenoceptors with a limited number of cysteines available for chemical derivatization were purified, site‐selectively labeled with the conformationally sensitive, cysteine‐reactive fluorophore IANBD and analyzed by fluorescence spectroscopy. Like the wild‐type receptor, mutant receptors containing 125Cys and/or 285Cys showed an agonist‐induced decrease in fluorescence, while no agonist‐induced response was observed in a receptor where these two cysteines were mutated. These data suggest that IANBD bound to 125Cys and 285Cys are exposed to a more polar environment upon agonist binding, and indicate that movements of transmembrane segments III and VI are involved in activation of G protein‐coupled receptors.

Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins

The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose–neopentyl glycol (MNG) amphiphile family show favorable behavior relative to conventional detergents, as manifested in multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.

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.

How curved membranes recruit amphipathic helices and protein anchoring motifs

Lipids and several specialized proteins are thought to be able to sense the curvature of membranes (MC). Here we used quantitative fluorescence microscopy to measure curvature-selective binding of amphipathic motifs on single liposomes 50-700 nm in diameter. Our results revealed that sensing is predominantly mediated by a higher density of binding sites on curved membranes instead of higher affinity. We proposed a model based on curvature-induced defects in lipid packing that related these findings to lipid sorting and accurately predicted the existence of a new ubiquitous class of curvature sensors: membrane-anchored proteins. The fact that unrelated structural motifs such as alpha-helices and alkyl chains sense MC led us to propose that MC sensing is a generic property of curved membranes rather than a property of the anchoring molecules. We therefore anticipate that MC will promote the redistribution of proteins that are anchored in membranes through other types of hydrophobic moieties.

Delineation of an endogenous zinc‐binding site in the human dopamine transporter

The molecular basis for substrate translocation in the Na+/Cl−‐dependent neurotransmitter transporters remains elusive. Here we report novel insight into the translocation mechanism by delineation of an endogenous Zn2+‐binding site in the human dopamine transporter (hDAT). In micromolar concentrations, Zn2+ was found to act as a potent, non‐competitive blocker of dopamine uptake in COS cells expressing hDAT. In contrast, binding of the cocaine analogue, WIN 35,428, was markedly potentiated by Zn2+. Surprisingly, these effects were not observed in the closely related human norepinephrine transporter (hNET). A single non‐conserved histidine residue (His193) in the large second extracellular loop (ECL2) of hDAT was discovered to be responsible for this difference. Thus, Zn2+ modulation could be conveyed to hNET by mutational transfer of only this residue. His375 conserved between hDAT and hNET, present in the fourth extracellular loop (ECL4) at the top of transmembrane segment VII, was identified as a second major coordinate for Zn2+ binding. These data provide evidence for spatial proximity between His193 and His375 in hDAT, representing the first experimentally demonstrated proximity relationship in an Na+/Cl−‐dependent transporter. Since Zn2+ did not prevent dopamine binding, but inhibited dopamine translocation, our data suggest that by constraining movements of ECL2 and ECL4, Zn2+ can restrict a conformational change critical for the transport process.

Calmodulin Kinase II Interacts with the Dopamine Transporter C Terminus to Regulate Amphetamine-Induced Reverse Transport

Efflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIalpha bound to the distal C terminus of DAT and colocalized with DAT in dopaminergic neurons. CaMKIIalpha stimulated dopamine efflux via DAT in response to amphetamine in heterologous cells and in dopaminergic neurons. CaMKIIalpha phosphorylated serines in the distal N terminus of DAT in vitro, and mutation of these serines eliminated the stimulatory effects of CaMKIIalpha. A mutation of the DAT C terminus impairing CaMKIIalpha binding also impaired amphetamine-induced dopamine efflux. An in vivo role for CaMKII was supported by chronoamperometry measurements showing reduced amphetamine-induced dopamine efflux in response to the CaMKII inhibitor KN93. Our data suggest that CaMKIIalpha binding to the DAT C terminus facilitates phosphorylation of the DAT N terminus and mediates amphetamine-induced dopamine efflux.

Amphipathic motifs in BAR domains are essential for membrane curvature sensing

BAR (Bin/Amphiphysin/Rvs) domains and amphipathic α‐helices (AHs) are believed to be sensors of membrane curvature thus facilitating the assembly of protein complexes on curved membranes. Here, we used quantitative fluorescence microscopy to compare the binding of both motifs on single nanosized liposomes of different diameters and therefore membrane curvature. Characterization of members of the three BAR domain families showed surprisingly that the crescent‐shaped BAR dimer with its positively charged concave face is not able to sense membrane curvature. Mutagenesis on BAR domains showed that membrane curvature sensing critically depends on the N‐terminal AH and furthermore that BAR domains sense membrane curvature through hydrophobic insertion in lipid packing defects and not through electrostatics. Consequently, amphipathic motifs, such as AHs, that are often associated with BAR domains emerge as an important means for a protein to sense membrane curvature. Measurements on single liposomes allowed us to document heterogeneous binding behaviour within the ensemble and quantify the influence of liposome polydispersity on bulk membrane curvature sensing experiments. The latter results suggest that bulk liposome‐binding experiments should be interpreted with great caution.