Ligand-Dependent Modulation of G Protein Conformation Alters Drug Efficacy.

G protein-coupled receptor (GPCR) signaling, mediated by hetero-trimeric G proteins, can be differentially controlled by agonists. At a molecular level, this is thought to occur principally via stabilization of distinct receptor conformations by individual ligands. These distinct conformations control subsequent recruitment of transducer and effector proteins. Here, we report that ligand efficacy at the calcitonin GPCR (CTR) is also correlated with ligand-dependent alterations to G protein conformation. We observe ligand-dependent differences in the sensitivity of the G protein ternary complex to disruption by GTP, due to conformational differences in the receptor-bound G protein hetero-trimer. This results in divergent agonist-dependent receptor-residency times for the hetero-trimeric G protein and different accumulation rates for downstream second messengers. This study demonstrates that factors influencing efficacy extend beyond receptor conformation(s) and expands understanding of the molecular basis for how G proteins control/influence efficacy. This has important implications for the mechanisms that underlie ligand-mediated biased agonism. VIDEO ABSTRACT.

G protein-coupled receptor interactions and modification of signalling involving the ghrelin receptor, GHSR1a.

The growth hormone secretagogue receptor 1a (GHSR1a) is intriguing because of its potential as a therapeutic target and its diverse molecular interactions. Initial studies of the receptor focused on the potential therapeutic ability for growth hormone (GH) release to reduce wasting in aging individuals, as well as food intake regulation for treatment of cachexia. Known roles of GHSR1a now extend to regulation of neurogenesis, learning and memory, gastrointestinal motility, glucose/lipid metabolism, the cardiovascular system, neuronal protection, motivational salience, and hedonic feeding. Ghrelin, the endogenous agonist of GHSR1a, is primarily located in the stomach and is absent from the central nervous system (CNS), including the spinal cord. However, ghrelin in the circulation does have access to a small number of CNS sites, including the arcuate nucleus, which is important in feeding control. At some sites, such as at somatotrophs, GHSR1a has high constitutive activity. Typically, ghrelin-dependent and constitutive GHSR1a activation occurs via Gαq/11 pathways. In vitro and in vivo data suggest that GHSR1a heterodimerises with multiple G protein-coupled receptors (GPCRs), including dopamine D1 and D2, serotonin 2C, orexin, oxytocin and melanocortin 3 receptors (MCR3), as well as the MCR3 accessory protein, MRAP2, providing possible mechanisms for its many physiological effects. In all cases, the receptor interaction changes downstream signalling and the responses to receptor agonists. This review discusses the signalling mechanisms of GHSR1a alone and in combination with other GPCRs, and explores the physiological consequences of GHSR1a coupling with other GPCRs.

Conformational Transitions and the Activation of Heterotrimeric G Proteins by G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) are particularly attractive targets for therapeutic pharmaceuticals. This is because they are involved in almost all facets of physiology, in many pathophysiological processes, they are tractable due to their cell surface location, and can exhibit highly textured pharmacology. While the development of new drugs does not require the molecular details of the mechanism of activity for a particular target, there has been increasing interest in the GPCR field in these details. In part, this has come with the recognition that differential activity at a particular target might be a way in which to leverage drug activity, either through manipulation of efficacy or through differential coupling (signaling bias). To this end, the past few years have seen a number of publications that have specifically attempted to address one or more aspects of the molecular reaction pathway, leading to activation of heterotrimeric G proteins by GPCRs.

Beyond mere markers: functions for CD34 family of sialomucins in hematopoiesis.

CD34, podocalyxin, and endoglycan are members of a family of single-pass transmembrane proteins that show distinct expression on early hematopoietic precursors and vascular-associated tissue. In spite of the fact that the expression of CD34 on these early progenitors has been known for over 20 yr and used clinically in hematopoietic stem cell transplantation for more than 15 yr, little is known about its exact role or function. More recently, CD34 expression has been shown to distinguish activated early progenitors from quiescent cells. With the subsequent identification of podocalyxin and endoglycan as related family members also expressed on early progenitor cells, attention is slowly shifting toward understanding how these molecules might contribute to progenitor function and behavior. In this review we examine the existing evidence and propose testable models to reveal the importance of these molecules for stem and progenitor cell function.

A simple method to generate stable cell lines for the analysis of transient protein-protein interactions.

Transient protein-protein interactions form the basis of signal transduction pathways in addition to many other biological processes. One tool for studying these interactions is bioluminescence resonance energy transfer (BRET). This technique has been widely applied to study signaling pathways, in particular those initiated by G protein-coupled receptors (GPCRs). These assays are routinely performed using transient transfection, a technique that has limitations in terms of assay cost and variability, overexpression of interacting proteins, vector uptake limited to cycling cells, and non-homogenous expression across cells within the assay. To address these issues, we developed bicistronic vectors for use with Life Technology's Gateway and flpIN systems. These vectors provide a means to generate isogenic cell lines for comparison of interacting proteins. They have the advantage of stable, single copy, isogenic, homogeneous expression with low inter-assay variation. We demonstrate their utility by assessing ligand-induced interactions between GPCRs and arrestin proteins.