David C. New

Molecular mechanisms mediating the G protein-coupled receptor regulation of cell cycle progression

G protein-coupled receptors are key regulators of cellular communication, mediating the efficient coordination of a cells responses to extracellular stimuli. When stimulated these receptors modulate the activity of a wide range of intracellular signalling pathways that facilitate the ordered development, growth and reproduction of the organism. There is now a growing body of evidence examining the mechanisms by which G protein-coupled receptors are able to regulate the expression, activity, localization and stability of cell cycle regulatory proteins that either promote or inhibit the initiation of DNA synthesis. In this review, we will detail the intracellular pathways that mediate the G protein-coupled receptor regulation of cellular proliferation, specifically the progression from the G1 phase to the S phase of the cell cycle.

The ORL1 receptor: molecular pharmacology and signalling mechanisms.

The cloning of the opioid-receptor-like 1 (ORL1) receptor and the identification of nociceptin as its endogenous agonist have revealed a new G-protein-coupled receptor signalling system. The structural and functional homology of ORL1 to the opioid receptor systems has posed a number of challenges in understanding the often competing physiological responses elicited by these G-protein-coupled receptors. Thus, this review will attempt to summarize recent research by many groups that has revealed numerous subtleties of the ORL1 receptor and its signalling pathways, as well as document the efforts to produce high-affinity selective ligands for the ORL1 receptor that may be of value as research and therapeutic tools.

G protein-coupled receptor-induced Akt activity in cellular proliferation and apoptosis

Akt (also known as protein kinase B) plays an integral role in many intracellular signaling pathways activated by a diverse array of extracellular signals that target several different classes of membrane‐bound receptors. Akt plays a particularly prominent part in signaling networks that result in the modulation of cellular proliferation, apoptosis and survival. Thus, the overexpression of Akt subtypes has been measured in a number of cancer types, and dominant‐negative forms of Akt can trigger apoptosis and reduce the survival of cancer cells. G protein‐coupled receptors act as cell‐surface detectors for a diverse spectrum of biological signals and are able to activate or inhibit Akt via several direct and indirect means. In this review, we shall document how G protein‐coupled receptors are able to control Akt activity and examine the resulting biochemical and physiological changes, with particular emphasis on cellular proliferation, apoptosis and survival.

Differential chemokine activation of CC chemokine receptor 1‐regulated pathways: ligand selective activation of Gα 14‐coupled pathways

Chemokines regulate the chemotaxis, development, and differentiation of many cell types enabling the regulation of routine immunosurveillance and immunological adaptation. CC chemokine receptor 1 (CCR1) is the target of 11 chemokines. This promiscuity of receptor‐ligand interactions and the potential for functional redundancy has led us to investigate the selective activation of CCR1‐coupled pathways by known CCR1 agonists. Chemokines leukotactin‐1, macrophage inflammatory protein (MIP)‐1α, monocyte chemotactic peptide (MCP)‐3, RANTES, and MIP‐1δ all inhibited adenylyl cyclase activity in cells transiently transfected with CCR1. In contrast, only MIP‐1δ was unable to signal via G14‐, G16‐ or chimeric 16z44‐coupled pathways. In a stable cell line expressing CCR1 and Gα14, all of these five chemokines along with hemofiltrate CC chemokine (HCC)‐1 and myeloid progenitor inhibitory factor (MPIF)‐1 were able to stimulate Gi/o‐coupled pathways, but MIP‐1δ, HCC‐1 and MPIF‐1 were unable to activate G14‐mediated stimulation of phospholipase Cβ activity. In addition, MIP‐1δ was unable to promote the phosphorylation of extracellular signal‐regulated kinase and c‐Jun N‐terminal kinase. This suggests that different chemokines are able to selectively activate CCR1‐coupled pathways, probably because of differentintrinsic ligand efficacies. CCR1 and Gα14 or Gα16 are co‐expressed in several cell types and we hypothesize that selective activation of chemokine receptors provides a mechanism by which chemokines are able to fine‐tune intracellular signaling pathways.

The Evidence for G-Protein-Coupled Receptors and Heterotrimeric G Proteins in Protozoa and Ancestral Metazoa

In higher eukaryotes G-protein-coupled signal transduction pathways are a common mechanism used to detect an extracellular message and transmit a signal, via a membrane-bound receptor and a heterotrimeric G protein, to second messenger producing enzymes and effector proteins. The techniques used to identify components of these pathways are increasingly being applied to protozoa and ancestral metazoa. Many of the organisms studied do seem to express functional homologues of those found in higher eukaryotes and increasingly genes encoding these proteins are being cloned. Sequence analysis of the isolated α-subunits of heterotrimeric G proteins shows that these proteins have extensive homology to their mammalian counterparts, and often show absolute sequence identity in functionally significant regions. The receptor clones isolated clearly establish that protozoa and early metazoa express proteins with seven transmembrane spanning domains. Comparisons with mammalian receptors indicate that these proteins are likely to be regulated by phosphorylation and dephosphorylation events, although the pathways which control these are yet to be identified. The postulated regulatory mechanisms and the number of homologous clones isolated from some protozoa suggest that a highly regulated system of transmembrane signalling appeared at a relatively early stage in evolution.

GABAB heterodimeric receptors promote Ca2+ influx via store-operated channels in rat cortical neurons and transfected chinese hamster ovary cells

The GABAB receptors are generally considered to be classical Gi-coupled receptors that lack the ability to mobilize intracellular Ca2+ without the aid of promiscuous G proteins. Here, we report the ability of GABAB receptors to promote calcium influx into primary cultures of rat cortical neurons and transfected Chinese hamster ovary cells. Chinese hamster ovary cells were transfected with GABAB1(a) or GABAB1(b) subunits along with GABAB2 subunits. In experiments using the fluorometric imaging plate reader platform, GABA and selective agonists promoted increases in intracellular Ca2+ levels in transfected Chinese hamster ovary cells and cortical neurons with the expected order of potency. These effects were fully antagonized by selective GABAB receptor antagonists. To investigate the intracellular pathways responsible for mediating these effects we employed several pharmacological inhibitors. Pertussis toxin abolished GABAB mediated Ca2+ increases, as did the phospholipase Cbeta inhibitor U73122. Inhibitor 2-aminethoxydiphenyl borane acts as an antagonist at inositol 1,4,5-trisphosphate receptors and at store-operated channels. In all cell types, 2-aminethoxydiphenyl borane prevented Ca2+ mobilization. The selective store-operated channel inhibitor 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride prevented increases in intracellular Ca2+ levels as did performing the assays in Ca2+ free buffers. In conclusion, GABAB receptors expressed in Chinese hamster ovary cells and endogenously expressed in rat cortical neurons promote Ca2+ entry into the cell via the activation of store-operated channels, using a mechanism that is dependent on Gi/o heterotrimeric proteins and phospholipase Cbeta. These findings suggest that the neuronal effects mediated by GABAB receptors may, in part, rely on the receptors ability to promote Ca2+ influx.

G Protein-Linked Effector and Second Messenger Systems Involved in Melatonin Signal Transduction

The isolation and characterization of multiple melatonin receptors in a wide range of tissues and cells signifies the functional diversity of melatonin. In different cellular environments, melatonin can regulate distinct second messengers or even positively or negatively regulate the same signal transduction pathway. The capacity by which melatonin receptors modulate the activities of various effector molecules is determined by the complement of signaling components present in any particular cell type. The specific interactions between many signaling molecules have been discerned in an increasing number of cellular systems and this information is being used to explain the physiological actions of melatonin. This review will attempt to summarize recent research by many groups that has revealed numerous subtleties of the melatonin-coupled signaling pathways.

BML-190 and AM251 act as inverse agonists at the human cannabinoid CB2 receptor: signalling via cAMP and inositol phosphates

The aminoalkylindole BML‐190 and diarylpyrazole AM251 ligands have previously been shown to bind to cannabinoid CB2 and CB1 receptors, respectively. In HEK‐293 cells stably expressing the human CB2 receptor, BML‐190 and AM251 potentiated the forskolin‐stimulated accumulation of cAMP. Moreover, the CB2 receptor can interact productively with 16z44, a promiscuous Gα16/z chimera. BML‐190 and AM251 reduce the basal levels of inositol phosphate production in cells expressing the CB2 receptor and 16z44. These results demonstrate that BML‐190 and AM251 act as inverse agonists at the human CB2 receptor acting via Gαi/o and Gαq family‐coupled pathways.

Synthesis of substituted N-[3-(3-methoxyphenyl)propyl] amides as highly potent MT2-selective melatonin ligands

A series of substituted N-[3-(3-methoxyphenyl)propyl] amides were synthesized and their binding affinities towards human melatonin MT(1) and MT(2) receptors were evaluated. It was discovered that a benzyloxyl substituent incorporated at C6 position of the 3-methoxyphenyl ring dramatically enhanced the MT(2) binding affinity and at the same time decreased MT(1) binding affinity.

Constitutive activation of the opioid receptor-like (ORL1) receptor by mutation of Asn133 to tryptophan in the third transmembrane region.

We have introduced a series of point mutations into the human opioid receptor‐like (ORL1) receptor and characterized them for their ability to constitutively activate G protein‐coupled receptor signalling pathways. Among the 12 mutants generated, mutation at Asn133 (N133W) gave increased basal signalling through three separate pathways. N133W increased the basal activity of G14‐ and G16‐dependent pathways by two‐ to three‐fold. The constitutive activity of the mutant was confirmed by the finding that the enhanced activity is dependent on the level of receptor expression. In HEK‐293 cells stably expressing N133W, signalling through Gi/o‐dependent pathways was also observed. Radioligand binding studies revealed that the affinity for nociceptin of the wild‐type ORL1 receptor and the N133W mutant do not differ significantly, suggesting that the ligand binding and signalling functions of constitutively active mutants of G protein‐coupled receptors are not necessarily intrinsically linked. In conclusion, our results demonstrate that a mutation in the third transmembrane domain is able to increase the basal signalling activity of the human ORL1 receptor.