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The movements of transmembrane segments (TMs) 3 and 6 at the cytoplasmic side of the membrane play an important role in the activation of G-protein-coupled receptors. Here we provide evidence for the existence of an ionic lock that constrains the relative mobility of the cytoplasmic ends of TM3 and TM6 in the inactive state of the ␤ 2 -adrenergic receptor. We propose that the highly conserved Arg-131 3.50 at the cytoplasmic end of TM3 interacts both with the adjacent Asp-130 3.49 and with Glu-268 6.30 at the cytoplasmic end of TM6. Such a network of ionic interactions has now been directly supported by the high-resolution structure of the inactive state of rhodopsin. We hypothesized that the network of interactions would serve to constrain the receptor in the inactive state, and the release of this ionic lock could be a key step in receptor activation. To test this hypothesis, we made charge-neutralizing mutations of Glu-268 The majority of hormones and neurotransmitters exerts its cellular effects by activating cell surface receptors belonging to the superfamily of G-protein-coupled receptors (GPCRs) 1 (1-3). The ␤ 2 -adrenergic receptor (␤ 2 AR) belongs to the subfamily of rhodopsin-like receptors and has been used as a prototype GPCR in numerous studies (1-3). Low-resolution structures of rhodopsin, resolved by Schertler and co-workers (4, 5), have demonstrated the presence of seven membrane-spanning ␣-helical segments and have provided important insights into the organization of the transmembrane bundle, allowing the development of tertiary structure models of GPCRs (6 -8). Importantly, a high-resolution structure of rhodopsin has now become available (9) making it possible to consider the functional roles of individual side chains from the perspective of an atomic resolution structure of a homologous GPCR.Understanding the function of GPCRs at a molecular level requires an understanding of how agonist binding to the receptor is converted into receptor activation (3). Studies based on EPR spectroscopy, fluorescence spectroscopy, alterations in cysteine accessibility, and engineering of metal-binding sites have altogether pointed to a key role for conformational changes of . The molecular mechanisms that underlie the movements of TM3 and TM6 and govern the transition of the receptor between its inactive and active states have nonetheless remained unclear. It has been suggested that the protonation of the aspartic acid in the highly conserved (D/E)RY motif at the cytoplasmic side of TM3 leads to a release of constraining intramolecular interactions, thereby resulting in the movements of TM3 and TM6 and a conversion of the receptor to the active state (7,14,16). This hypothesis has been supported by the observation that charge-neutralizing mutations of the aspartic acid (or glutamic acid) in TM3 lead to increased agonist-independent activation of a number of GPCRs (7,14,17,18). Moreover, direct evidence has been obtained indicating that the photoactivation of rhodopsin is accompanied by the uptake of a proton b...

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.

G protein-coupled, seven-transmembrane segment receptors (GPCRs or 7TM receptors), with more than 1000 different members, comprise the largest superfamily of proteins in the body. Since the cloning of the first receptors more than a decade ago, extensive experimental work has uncovered multiple aspects of their function and challenged many traditional paradigms. However, it is only recently that we are beginning to gain insight into some of the most fundamental questions in the molecular function of this class of receptors. How can, for example, so many chemically diverse hormones, neurotransmitters, and other signaling molecules activate receptors believed to share a similar overall tertiary structure? What is the nature of the physical changes linking agonist binding to receptor activation and subsequent transduction of the signal to the associated G protein on the cytoplasmic side of the membrane and to other putative signaling pathways? The goal of the present review is to specifically address these questions as well as to depict the current awareness about GPCR structure-function relationships in general.

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 [ 3 H]CFT in the transporter, either by cross-linking engineered cysteines or with an engineered Zn 2+ -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.Correspondence should be addressed to U.G. (E-mail: gether@sund.ku.dk). Note: Supplementary information is available on the Nature Neuroscience website. AUTHOR CONTRIBUTIONST.B. designed and performed the computational experiments, analyzed the data and wrote the manuscript draft together with C.J.L. J.K. generated mutants, carried out pharmacological analyses and contributed to the data analysis. M.L.B. and K.R. generated mutants and carried out pharmacological analyses. L.S. contributed to the computational experiments and manuscript refinement. L.G. participated in the design and performance of the computational experiments. A.H.N. contributed with ideas, benztropine analogues and provided expertise in the pharmacology and medicinal chemistry of DAT inhibitors. J.A.J. contributed with ideas and to the design of experiments and writing of the manuscript. H.W. directed the design and performance of the modeling and computational experiments, participated in data analysis and contributed to writing the manuscript. U.G. supervised the project together with C.J.L., designed experiments, analyzed data and wrote the final manuscript. C.J.L. supervised the project together with U.G., designed experiments, generated mutants, performed pharmacological experiments, analyzed data and wrote the manuscript draft together with T.B.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ NIH Public Access Author ManuscriptNat Neurosci. Author manuscript; available in PMC 2009 July 1. Published in final edited form as:Nat Neurosci. 2008 July ; 11(7): 780-789. doi:10.1038/nn.2146. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptCocaine is an alkaloid derived from the Peruvian Erythroxylon coca plant and has been used as a stimulant for centuries 1 . Today, cocaine is widely abused, especially in the western hemisphere, causing major socioeconomic burdens through increased medical expenses, lost earnings and increased crime 2 . Nonetheless, the molecular mechanisms underlying cocaine's pharmacology and abuse ...