Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep integral membrane proteins (MPs) water soluble. In this review, we discuss their structure and solution behavior; the way they associate with MPs; and the structure, dynamics, and solution properties of the resulting complexes. All MPs tested to date form water-soluble complexes with APols, and their biochemical stability is in general greatly improved compared with MPs in detergent solutions. The functionality and ligand-binding properties of APol-trapped MPs are reviewed, and the mechanisms by which APols stabilize MPs are discussed. Applications of APols include MP folding and cell-free synthesis, structural studies by NMR, electron microscopy and X-ray diffraction, APol-mediated immobilization of MPs onto solid supports, proteomics, delivery of MPs to preexisting membranes, and vaccine formulation.

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To maintain homeostasis, hypothalamic neurons in the arcuate nucleus must dynamically sense and integrate a multitude of peripheral signals. Blood-borne molecules must therefore be able to circumvent the tightly sealed vasculature of the blood-brain barrier to rapidly access their target neurons. However, how information encoded by circulating appetite-modifying hormones is conveyed to central hypothalamic neurons remains largely unexplored. Using in vivo multiphoton microscopy together with fluorescently labeled ligands, we demonstrate that circulating ghrelin, a versatile regulator of energy expenditure and feeding behavior, rapidly binds neurons in the vicinity of fenestrated capillaries, and that the number of labeled cell bodies varies with feeding status. Thus, by virtue of its vascular connections, the hypothalamus is able to directly sense peripheral signals, modifying energy status accordingly.hormone diffusion | in vivo imaging | median eminence | metabolism C ontinuous integration of peripheral signals by neurons belonging to the arcuate nucleus of the hypothalamus (ARH) is critical for central regulation of energy balance and neuroendocrine function (1). To dynamically report alterations to homeostasis and ensure an appropriate neuronal response, blood-borne factors such as hormones must rapidly access the central nervous system (CNS). This is particularly evident in the case of food intake, which is regulated by a plethora of circulating satiety signals (2) whose levels fluctuate in an ultradian manner. Despite this, it remains unclear how key energy status-signaling hormones such as ghrelin can be rapidly sensed by target neurons to alter feeding responses (3). Elucidation of the mechanisms underlying molecule entry into the brain is important for understanding not only normal maintenance of homeostasis but also how this is perturbed during common pathologies such as obesity and diabetes (4, 5).Although molecule transport mechanisms within the ARH are poorly characterized, they likely assume one of two forms. First, chronic feedback may be accomplished by uptake of circulating molecules into the ARH via saturable receptor-mediated transport at the level of the choroid plexus and/or bloodbrain barrier (BBB) (6-9). Second, the ARH is morphologically located in close apposition to the median eminence (ME), a circumventricular organ composed of fenestrated capillaries. Because these vessels project toward the ventromedial ARH (vmARH), they could represent a direct vascular input for passive diffusion of peripheral molecules into the hypothalamus (10-13). So far, study of the functional importance of fenestrated capillaries in molecule entry into the metabolic brain has been impeded by lack of appropriate tools.To evaluate the role of fenestrated ME/ARH capillaries in rapid detection of peripheral signals by the hypothalamus, we used a recently developed in vivo imaging approach to visualize in real time the extravasation of fluorescent molecules (14). Ghrelin was chosen as a candidate hormone because i...

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs.

The ghrelin receptor or growth hormone secretagogue receptor (GHSR) is a G-protein-coupled receptor that controls growth hormone and insulin secretion, food intake, and reward-seeking behaviors. Liver-expressed antimicrobial peptide 2 (LEAP2) was recently described as an endogenous antagonist of GHSR. Here, we present a study aimed at delineating the structural determinants required for LEAP2 activity toward GHSR. We demonstrate that the entire sequence of LEAP2 is not necessary for its actions. Indeed, the N-terminal part alone confers receptor binding and activity to LEAP2. We found that both LEAP2 and its N-terminal part behave as inverse agonists of GHSR and as competitive antagonists of ghrelin-induced inositol phosphate production and calcium mobilization. Accordingly, the N-terminal region of LEAP2 is able to inhibit ghrelin-induced food intake in mice. These data demonstrate an unexpected pharmacological activity for LEAP2 that is likely to have an important role in the control of ghrelin response under normal and pathological conditions.

The eight metabotropic glutamate receptors (mGluRs) are key modulators of synaptic transmission and are considered promising targets for the treatment of various brain disorders. Whereas glutamate acts at a large extracellular domain, allosteric modulators have been identified that bind to the seven transmembrane domain (7TM) of these dimeric G-protein-coupled receptors (GPCRs). We show here that the dimeric organization of mGluRs is required for the modulation of active and inactive states of the 7TM by agonists, but is not necessary for G-protein activation. Monomeric mGlu2, either as an isolated 7TM or in full-length, purified and reconstituted into nanodiscs, couples to G proteins upon direct activation by a positive allosteric modulator. However, only a reconstituted full-length dimeric mGlu2 activates G protein upon glutamate binding, suggesting that dimerization is required for glutamate induced activation. These data show that, even for such well characterized GPCR dimers like mGluR2, a single 7TM is sufficient for G-protein coupling. Despite this observation, the necessity of dimeric architecture for signaling induced by the endogenous ligand glutamate confirms that the central core of signaling complex is dimeric.M etabotropic glutamate receptors (mGluRs) play key roles in the modulation of both excitatory and inhibitory synapses in the brain. These eight G-protein-coupled receptors (GPCRs) represent major targets for pharmaceutical companies in search of new treatments for a variety of neurological and psychiatric disorders (1-3). These receptors are part of the class C GPCR family that also includes the GABA B , calcium sensing, and sweet and umami taste receptors, which are all major targets for drug development (4).The structural complexity of class C GPCRs, compared with rhodopsin-like class A GPCRs, offers multiple possibilities in designing molecules that modulate their activity. Not only are mGluRs strict constitutive dimers (5, 6), but each protomer is composed of several domains (7,8). Agonists bind in a bilobate venus fly-trap domain (VFT) (9), which is linked through a cysteine-rich domain (CRD) to the heptahelical transmembrane domain (7TM) that is responsible for G-protein activation (7). The 7TM is the target of a number of synthetic compounds acting either as negative or positive allosteric modulators (NAMs and PAMs, respectively). Given their ability to finely tune endogenous signaling, such compounds present exciting opportunities for drug development (10).The functional mechanism of such a complex machine remains to be characterized, although some critical steps have been well documented. Previous studies have shown that receptor activation results from the closure of the VFT upon agonist binding (9,(11)(12)(13)(14)(15). This conformational change in the extracellular domain is coupled to a conformational change in the intracellular side of at least one 7TM that is responsible for G-protein coupling (16)(17)(18)(19). The mechanism for allosteric communication between the VFT and 7TM ...

Serotonin 5-HT 4(a) receptor, a G-protein-coupled receptor (GPCR), was produced as a functional isolated protein using Escherichia coli as an expression system. The isolated receptor was characterized at the molecular level by circular dichroism (CD) and steady-state fluorescence. A specific change in the near-UV CD band associated with the GPCR disulfide bond connecting the third transmembrane domain to the second extracellular loop (e2) was observed upon agonist binding to the purified receptor. This is a direct experimental evidence for a change in the conformation of the e2 loop upon receptor activation. Different variations were obtained depending whether the ligand was an agonist (partial or full) or an inverse agonist. In contrast, antagonist binding did not induce any variation. These observations provide a first direct evidence for the fact that free (or antagonist-occupied), active (partial-or full agonist-occupied) and silent (inverse agonist-occupied) states of the receptor involve different arrangements of the e2 loop. Finally, ligand-induced changes in the fluorescence emission profile of the purified receptor confirmed that the partial agonist stabilized a single, welldefined, conformational state and not a mixture of different states. This result is of particular interest in a pharmacological perspective since it directly demonstrates that the efficacy of a drug is likely due to the stabilization of a ligand-specific state rather than selection of a mixture of different conformational states of the receptor.G-protein-coupled receptors are versatile biological sensors. They are responsible for the majority of cellular responses to hormones and neurotransmitters, as well as sight, smell, and taste senses (1, 2). Signal transduction is specifically associated with GPCR 1 activation. Although significant progress has been made within the last few years in dissecting GPCR-mediated signal transduction pathways, understanding of the mechanisms underlying receptor activation is still hampered by the lack of information at the molecular level (3,4). This is largely due to the fact that very few expression systems have proven satisfactory for producing these receptors in a functional state and with sufficient yields for biophysical studies to be carried out (5-7). Most of the systems that have been developed to elucidate the mechanism of GPCR activation therefore essentially rely on the use of purified rhodopsin and ␤ 2 -adrenergic receptor (3,4,8). Interestingly, most of the results obtained so far report on the conformational events occurring at the level of the cytoplasmic side of the receptors. In contrast, few reports give indications on the possible conformational rearrangements certainly occurring in the extracellular part of the receptor, in particular in the extracellular loops.Several models have been developed to conceptualize the mechanisms of activation (9, 10). The two-state model and the extended ternary model assumes that the receptor exists in an equilibrium between a resting state (R) and...

G-protein-coupled receptors (GPCRs) are key players in cell communication. Although long considered as monomeric, it now appears that these heptahelical proteins can form homo-or heterodimers. Here, we analyzed the conformational changes in each subunit of a receptor dimer resulting from agonist binding to either one or both subunits by measuring the fluorescent properties of a leukotriene B 4 receptor dimer with a single 5-hydroxytryptophan-labeled protomer. We show that a receptor dimer with only a single agonist-occupied subunit can trigger G-protein activation. We also show that the two subunits of the receptor dimer in the G-protein-coupled state differ in their conformation, even when both are liganded by the agonist. No such asymmetric conformational changes are observed in the absence of G-protein, indicating that the interaction of the G-protein with the receptor dimer brings specific constraints that prevent a symmetric functioning of this dimer. These data open new options for the differential signaling properties of GPCR dimers.