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As a G protein-coupled receptor, the extracellular [Ca.sup.2+]-sensing receptor (CaSR) responds to changes not only in extracellular [Ca.sup.2+], but also to many other ligands. CaSR has been found to be expressed in the hearts and cardiovascular system. In this study, we confirmed that CaSR is expressed in mouse cardiomyocytes and showed that it is predominantly localized in caveolae. The goal of this study was to investigate whether CaSR plays a cardioprotective role in ischemic preconditioning (IPC). Hearts from C57BL/6J mice (male, 12-16 wk) were perfused in the Langendorff mode and subjected to the following treatments: 1) control perfusion; 2) perfusion with a specific CaSR antagonist, NPS2143; 3) IPC (four cycles of 5 min of global ischemia and 5 min of reperfusion); or 4) perfusion with NPS2143 before and during IPC. Following these treatments, hearts were subjected to 20 min of no-flow global ischemia and 120 min of reperfusion. Compared with control, IPC significantly improved postischemic left ventricular functional recovery and reduced infarct size. Although NPS2143 perfusion alone did not change the hemodynamic function and did not change the extent of postischemic injury, NPS2143 treatment abolished cardioprotection of IPC. Through immunoblot analysis, it was demonstrated that IPC significantly increased the levels of phosphorylated ERK1/2, AKT, and GSK-3[beta], which were also prevented by NPS2143 treatment. Taken together, the distribution of CaSR in caveolae along with NPS2143-blockade of IPC-induced cardioprotective signaling suggest that the activation of CaSR during IPC is cardioprotective by a process involving caveolae. cardioprotection; caveolae; caveolin-1 doi: 10.1152/ajpheart.00373.2010.

The development of cardiac hypertrophy in response to increased hemodynamic load and neurohormonal stress is initially a compensatory response that may eventually lead to ventricular dilation and heart failure. Regulator of G protein signaling 5 (Rgs5) is a negative regulator of G protein-mediated signaling by inactivating G[alpha](q) and G[alpha](i), which mediate actions of most known vasoconstrictors. Previous studies have demonstrated that Rgs5 expresses among various cell types within mature heart and showed high levels of Rgs5 mRNA in monkey and human heart tissue by Northern blot analysis. However, the critical role of Rgs5 on cardiac remodeling remains unclear. To specifically determine the role of Rgs5 in pathological cardiac remodeling, we used transgenic mice with cardiac-specific overexpression of human Rgs5 gene and [Rgs5.sup.-/-] mice. Our results demonstrated that the transgenic mice were resistant to cardiac hypertrophy and fibrosis through inhibition of MEK-ERK1/2 signaling, whereas the [Rgs5.sup.-/-] mice displayed the opposite phenotype in response to pressure overload. These studies indicate that Rgs5 protein is a crucial component of the signaling pathway involved in cardiac remodeling and heart failure. ERK 1/2 | RGS5 | MEK 1/2 | signal transduction doi/ 10.1073/pnas.1008397107

The Toll-like receptor (TLR)4 receptor complex, TLR4/MD-2, plays an important role in the inflammatory response against lipopolysaccharide, a ubiquitous membrane component in Gram-negative bacteria. Ligand recognition by TLR4 initiates multiple intracellular signaling pathways, leading to production of proinflammatory mediators and type I IFN. Ligand interaction also leads to internalization of the surface receptor complex into lysosomes, leading to the degradation of TLR4 and the termination of LPS response. However, surface level of TLR4 receptor complex is maintained via continuous replenishment of TLR4 from intracellular compartments like Golgi and endosomes. Here we show that continuous replenishment of TLR4 from Golgi to plasma membrane is regulated by the small GTPase Rab10, which is essential for optimal macrophage activation following LPS stimulation. Expression of Rab10 is inducible by LPS. Blockade of Rab10 function leads to decreased membrane TLR4 expression and diminished production of inflammatory cytokines and interferons upon LPS stimulation. These findings suggest that Rab10 expression provides a mechanism to refine TLR4 signaling by regulating the trafficking rate of TLR4 onto the plasma membrane. In addition, we show that altered Rab10 expression in macrophages influences disease severity in an in vivo model of LPS-induced acute lung injury, suggesting Rab10 as a possible therapeutic target for human acute respiratory distress syndrome (ARDS). small GTPases | membrane trafficking | LPS | acute respiratory distress syndrome doi/ 10.1073/pnas.1009428107

Am J Physiol Heart Circ Physiol 298: H16-H23, 2010. First published October 30, 2009; doi: 10.1152/ajpheart.00588.2009.--Several studies have recently demonstrated that G protein-coupled receptor 30 (GPER) can directly bind to estrogen and mediate its action. We investigated the role and the mechanism of estrogen-induced cardio-protection after ischemia-reperfusion using a specific GPER agonist G1. Isolated hearts from male mice were perfused using Langendorff technique with oxygenated (95% [O.sub.2] and 5% C[O.sub.2]) Krebs Henseleit buffer (control), with G1 (1 [micro]M), and G1 (1 [micro]M) together with [Ca.sup.2+]acellular signal-regulated kinase (Erk) inhibitor PD-98059 (5 [micro]M). After 20 min of perfusion, hearts were subjected to 20 min global normothermic (37[degrees]C) ischemia followed by 40 min reperfusion. Cardiac function was measured, and myocardial necrosis was evaluated by triphenyltetrazolium chloride staining at the end of the reperfusion. Mitochondria were isolated after 10 min of reperfusion to assess the [Ca.sup.2+] load required to induce mitochondria permeability transition pore (mPTP) opening. Gl-treated hearts developed better functional recovery with higher rate pressure product (RPP, 6140 [+ or ] 264 vs. 2,640 [+ or ] 334 beats x mm[Hg.sup.-1] x [min.sup.-1,] P < 0.05). The infarct size decreased significantly in Gl-treated hearts (21 [+ or ] 2 vs. 46 [+ or ] 3%, P < 0.001), and the [Ca.sup.2+] load required to induce mPTP opening increased (2.4 [+ or ] 0.06 vs. 1.6 [+ or ] 0.11 [micro]M/mg mitochondrial protein, P < 0.05) compared with the controls. The protective effect of G1 was abolished in the presence of PD-98059 [RPP: 4,120 [+ or ] 46 beats x mm[Hg.sup.-1] x [min.sup.-1], infarct size: 53 [+ or ] 2%, and [Ca.sup.2+] retention capacity: 1.4 [+ or ] 0.11 [micro]M/mg mitochondrial protein (P < 0.05)]. These results suggest that GPER activation provides a cardioprotectire effect after ischemia-reperfusion by inhibiting the mPTP opening, and this effect is mediated by the Erk pathway. G protein-coupled receptor 30; mitochondrial permeability transition pore; cardioprotection; infarct size; heart function

The regulator of G protein signaling (RGS) proteins are a family of guanosine triphosphatase (GTPase)--accelerating proteins. We have discovered a novel function for RGS2 in the control of protein synthesis. RGS2 was found to bind to elF2Be (eukaryotic initiation factor 2B [epsilon] subunit) and inhibit the translation of messenger RNA (mRNA) into new protein. This effect was not observed for other RGS proteins tested. This novel function of RGS2 is distinct from its ability to regulate G protein--mediated signals and maps to a stretch of 37 amino acid residues within its conserved RGS domain. Moreover, RGS2 was capable of interfering with the elF2--elF2B GTPase cycle, which is a requisite step for the initiation of mRNA translation. Collectively, this study has identified a novel role for RGS2 in the control of protein synthesis that is independent of its established RGS domain function.

We have earlier shown that the treatment of A10 vascular smooth muscle cells with S-nitroso-N-acetyl-penicillamine (SNAP); nitric oxide donor (NO) for 24 h decreased the expression of natriuretic peptide receptor C (NPR-C) and adenylyl cyclase signaling. The present study was undertaken to examine the implication of different signaling mechanisms in a NO-induced response. The treatment of A10 vascular smooth muscle cells with SNAP decreased the expression of NPR-C and [G.sub.i][alpha] proteins in a time-dependent manner. The expression of [G.sub.i][alpha] proteins was decreased at 6 h, whereas the expression of NPR-C was attenuated at 2 h. The NPR-C-mediated inhibition of adenylyl cyclase was attenuated (~50%) after 2 h of treatment and was completely abolished after 6 h of treatment. The decreased expression of NPR-C and NPR-C-mediated attenuation of adenylyl cyclase after 2 h of treatment was reversed to control levels by PD-98059, a MEK inhibitor. SNAP also modulated the ERK1/2 phosphorylation in a time-dependent manner; an increase was observed up to 2 h, and, thereafter, the ERK1/2 phosphorylation was decreased. On the other hand, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and KT-5823 inhibitor of soluble guanylyl cyclase and protein kinase G, respectively, and Mn(III)tetrakis(4-benzoic acid)porphyrin, a scavenger of peroxynitrite, were unable to restore the SNAP-induced decreased expression of NPR-C protein and increased ERK1/2 phosphorylation to control levels. However, the decreased levels of phosphorylated ERK1/2 and [G.sub.i][alpha] proteins were restored to control levels by 8-bromo-cAMP. These results indicate that a temporal relationship follows between a NO-induced decreased expression of NPR-C and [G.sub.i][alpha] proteins. The decreased expression of NPR-C is mediated through cGMP-independent but MAPK-dependent pathway, whereas NO-induced decreased levels of cAMP may contribute to the decreased activation of MAPK and thereby decreased the expression of [G.sub.i][alpha] proteins. G proteins; mitogen-activated protein kinase

We outline a powerful method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli. For a mammalian G protein-coupled receptor, we arrived at a sequence with an order-of-magnitude increase in functional expression that still retains the biochemical properties of wild type. This mutant also shows enhanced heterologous expression in eukaryotes (12-fold in Pichia pastoris and 3-fold in HEK293T cells) and greater stability when solubilized and purified, indicating that the biophysical properties of the protein had been under the pressure of selection. These improvements arise from multiple small contributions, which would be difficult to assemble by rational design. In a second screen, we rapidly pinpointed a single amino acid substitution in wild type that abolishes antagonist binding while retaining agonist-binding affinity. These approaches may alleviate existing bottlenecks in structural studies of these targets by providing sufficient quantities of stable variants in defined conformational states. integral membrane proteins | protein engineering | protein folding

The cgtA gene, coding for the conserved G protein CgtA, is essential in bacteria. In contrast to a previous report, here we show by using genetic analysis that cgtA is essential in Vibrio cholerae even in a [DELTA]relA background. Depletion of CgtA affected the growth of V. cholerae and rendered the cells highly sensitive to the replication inhibitor hydroxyurea. Overexpression of V. cholerae CgtA caused distinct elongation of Escherichia coli cells. Deletion analysis indicated that the C-terminal end of CgtA plays a critical role in its proper function.

The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110[alpha], p110[beta], and p110[delta]) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110[alpha] and p110[delta] to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110[gamma], class IB PI3K lack SH2 domains and instead couple p110[gamma], to G protein-coupled receptors (GPCRs). Here, we show, using small molecule inhibitors with selectivity for p110[beta] and cells derived from a p110[beta]-deficient mouse line, that p110[beta] is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110[beta] and p110[gamma], contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110[beta] but not p110[gamma], p11[beta] mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110 [gamma], in these cells reduced the contribution of p110[beta] to GPCR signaling. Taken together, these data show that p110[beta] and p110[gamma], can couple redundantly to the same GPCR agonists, p110[beta], which shows a much broader tissue distribution than the leukocyte-restricted p110[gamma] could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110[gamma] expression is low or absent. gene targeting | signaling | tyrosine kinase | Akt | insulin

The formation of chimeric mRNAs is a strategy used by human cells to increase the complexity of their proteome, as revealed by the ENCODE project. Here, we use Saccharomyces cerevisiae to show a way by which trans-spliced mRNAs can be generated. We demonstrate that a pretRNA inserted into a premRNA context directs the splicing reaction precisely to the sites of the tRNA intron. A suppressor pretRNA gene was inserted, in cis, into the sequence encoding the third cytoplasmic loop of the Ste2 or Ste3 G protein-coupled receptor. The hybrid RNAs are spliced at the specific pretRNA splicing sites, releasing both functional tRNAs that suppress nonsense mutations and translatable mRNAs that activate the signal transduction pathway. The RNA molecules extracted from yeast cells were amplified by RT-PCR, and their sequences were determined, confirming the identity of the splice junctions. We then constructed two fusions between the premRNA sequence (STE2 or STE3) and the 5'- or 3'-pretRNA half, so that the two hybrid RNAs can associate with each other, in trans, through their tRNA halves. Splicing occurs at the predicted pretRNA sites, producing a chimeric STE3-STE2 receptor mRNA. RNA trans-splicing mediated by tRNA sequences, therefore, is a mechanism capable of producing new kinds of RNAs, which could code for novel proteins. ENCODE | G protein-coupled receptors | genomics | tRNA endonuclease

Author(s): Markus Brockmann [1]; Vincent A. Blomen [1]; Joppe Nieuwenhuis [1]; Elmer Stickel [1]; Matthijs Raaben [1]; Onno B. Bleijerveld [1]; A. F. Maarten Altelaar [1, 2]; Lucas T. Jae [...]

Ras has achieved notoriety as an oncogene aberrantly activated in multiple human tumors. Approximately 30% of all human tumors express an oncogenic form of this GTPase that is locked in an active conformation as a result of being insensitive to Ras GTPase-activating proteins (GAPs), proteins that normally regulate the inactivation of Ras by enhancing its intrinsic GTPase activity. Besides oncogenic mutations in Ras, signaling by wild-type Ras is also frequently deregulated in tumors through aberrant coupling to activated cell surface receptors. This indicates that alternative mechanisms of aberrant wild-type Ras activation may be involved in tumorigenesis. Here, we describe another mechanism through which aberrant Ras activation is achieved in human cancers. We have established that Ras GTPase-activating-like protein (RASAL), a [Ca.sup.2+]-regulated Ras GAP that decodes the frequency of [Ca.sup.2+] oscillations, is silenced through CpG methylation in multiple tumors. With the finding that ectopic expression of catalytically active RASAL leads to growth inhibition of these tumor cells by Ras inactivation, we have provided evidence that epigenetically silencing of this Ras GAP represents a mechanism of aberrant Ras activation in certain cancers. Our demonstration that RASAL constitutes a tumor suppressor gene has therefore further emphasized the importance of [Ca.sup.2+] in the regulation of Ras signaling and has established that deregulation of this pathway is an important step in Ras-mediated tumorigenesis. calcium | Ras GTPase-activating-like protein | tumorigenesis

Rab proteins are small GTPases that play important roles in transport of vesicle cargo and recruitment, association of motor and other proteins with vesicles, and docking and fusion of vesicles at defined locations. In vertebrates, > 75 Rab genes have been identified, some of which have been intensively studied for their roles in endosome and synaptic vesicle trafficking. Recent studies of the functions of certain Rab proteins have revealed specific roles in mediating developmental signal transduction. We have begun a systematic genetic study of the 33 Rab genes in Drosophila. Most of the fly proteins are clearly related to specific vertebrate proteins. We report here the creation of a set of transgenic fly lines that allow spatially and temporally regulated expression of Drosophila Rab proteins. We generated fluorescent proteintagged wild-type, dominant-negative, and constitutively active forms of 31 Drosophila Rab proteins. We describe Drosophila Rab expression patterns during embryogenesis, the subcellular localization of some Rab proteins, and comparisons of the localization of wild-type, dominant-negative, and constitutively active forms of selected Rab proteins. The high evolutionary conservation and low redundancy of Drosophila Rab proteins make these transgenic lines a useful tool kit for investigating Rab functions in vivo.

The advances in the mechanism of regulation and signaling and the roles in vivo of the GTPase family are discussed. Rho, Rac and Cdc42 are the best-characterized Rho/Rac subfamilies, which play important roles in different cellular processes.

G-proteins play critical roles in many cellular processes and are regulated by accessory proteins that modulate the nucleotide-bound state. Such proteins, including eukaryotic translation elongation factor 1A (eEF1A), are frequently reactivated by guanine nucleotide exchange factors (GEFs). In the yeast Saccharomyces cerevisiae, only the catalytic subunit of the GEF complex, eEF1B[alpha], is essential for viability. The requirement for the TEF5 gene encoding eEF1B[alpha] can be suppressed by the presence of excess substrate, eEF1A. These cells, however, have defects in growth and translation. Two independent unbiased screens performed to dissect the cause of these phenotypes yielded dominant suppressors that bypass the requirement for extra eEF1A. Surprisingly, all mutations are in the G-protein eEF1A and cluster in its GTP-binding domain. Five mutants were used to construct novel strains expressing only the eEF1A mutant at normal levels. These swains show no growth defects and little to no decreases in total translation, which raises questions as to the evolutionary expression of GEF complexity and other potential functions of this complex. The location of the mutations on the eEF1A-eEF1B[alpha] structure suggests that their mechanism of suppression may depend on effects on the conserved G-protein elements: the P-loop and NKXD nucleotide-binding element.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tips.2006.03.005 Byline: Lee S. Weinstein, Min Chen, Tao Xie, Jie Liu Abstract: Heterotrimeric G proteins couple receptors for diverse extracellular signals to effector enzymes or ion channels. Each G protein comprises a specific [alpha]-subunit and a tightly bound [beta][gamma] dimer. Several human disorders that result from genetic G-protein abnormalities involve the imprinted GNAS gene, which encodes G.sub.s[alpha], the ubiquitously expressed [alpha]-subunit that couples receptors to adenylyl cyclase and cAMP generation. Loss-of-function and gain-of-function mutations, in addition to imprinting defects, of this gene lead to diverse clinical phenotypes. Mutations of GNAT1 and GNAT2, which encode the retinal G proteins (transducins), are rare causes of specific congenital visual defects. Common polymorphisms of the GNAS and GNB3 (which encodes G[beta].sub.3) genes have been associated with multigenic disorders (e.g. hypertension and metabolic syndrome). To date, no other G proteins have been implicated directly in human disease. Author Affiliation: Metabolic Diseases Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA

Heterotrimeric G proteins couple receptors for diverse extracellular signals to effector enzymes or ion channels. Each G protein comprises a specific [alpha]-subunit and a tightly bound [beta][gamma] dimer. Several human disorders that result from genetic G-protein abnormalities involve the imprinted GNAS gene, which encodes [G.sub.s][alpha], the ubiquitously expressed [alpha]-subunit that couples receptors to adenylyl cyclase and cAMP generation. Loss-of-function and gain-of-function mutations, in addition to imprinting defects, of this gene lead to diverse clinical phenotypes. Mutations of GNAT1 and GNAT2, which encode the retinal G proteins (transducins), are rare causes of specific congenital visual defects. Common polymorphisms of the GNAS and GNB3 (which encodes G[[beta].sub.3]) genes have been associated with multigenic disorders (e.g. hypertension and metabolic syndrome). To date, no other G proteins have been implicated directly in human disease.

Maintenance of meiotic arrest is performed by the G-protein-coupled receptor GPR3 in mouse oocyte, instead of follicle cells. Implications for studying a broad range of other proteins in early embryonic development are discussed.

An early drug discovery approach focusing on gene families can benefit from strategies that exploit common signaling mechanisms to more effectively identify and characterize novel chemical lead structures. Multiplexing, defined [...]

Oocyte maturation (OM) is initiated in lower vertebrates and echinoderms when maturation-inducing substances (MIS) bind oocyte membrane receptors. This study tested the hypothesis that activation of a [G.sub.i] protein is necessary for MIS-mediated OM in spotted seatrout. Addition of MIS significantly decreased adenylyl cyclase activity in a steroid specific, pertussis toxin (PTX)-sensitive manner in oocyte membranes and microinjection of PTX into oocytes inhibited MIS-induced OM, suggesting the steroid activates a [G.sub.i] protein. MIS significantly increased [[sup.35]S]GTP[gamma]S binding to ovarian membranes, confirming that MIS receptor binding activates a G-protein, and immunoprecipitation studies showed the increased [[sup.35]S]GTP[gamma]S binding was associated with [G[[alpha].sub.i1-3] proteins. Radioligand binding studies in ovarian membranes using GTP[gamma]S and PTX demonstrated that the MIS binds a receptor coupled to a PTX-sensitive G-protein. This study provides the first direct evidence in a vertebrate model that MIS-induced activation of a [G.sub.i] protein is necessary for OM. These results support a mechanism of MIS action involving binding to a novel, G-protein coupled receptor and activation of an inhibitory G-protein, the most comprehensive and plausible model of MIS initiation of OM proposed to date. Keywords: Oocyte maturation; G-protein; Germinal vesicle breakdown; Membrane progestin receptor; Spotted seatrout

The vomeronasal organ of the accessory olfactory system detects pheromones in several vertebrate species. Recent studies of vomeronasal sensory neurons have shown that they express MHC molecules, which in the immune system help to discriminate self antigens from non-self antigens. These new findings, along with past research demonstrating MHC-based olfactory discrimination, suggest the exciting possibility that MHC molecules together with vomeronasal G-protein-coupled receptors play a role in distinguishing related individuals from unrelated ones based on pheromonal cues.

It is now ~5 years since the identification of the family of receptor activity modifying proteins (RAMPs). This finding revolutionized concepts of the pharmacology of G-protein-coupled receptors (GPCRs) and revealed that GPCR accessory proteins not only assist trafficking and folding but also define receptor type. Since the identification of RAMPs as modulators of the trafficking and properties of the calcitonin-receptor-like receptor, much work has focused on improving our understanding of the nature of RAMP--GPCR dimers, the extent to which they occur, and the consequence of this association. In this article, we review recent developments, including the identification of new receptor partners and novel roles for RAMPs.

There has been much interest in determining the molecular and cellular functions of hamartin and tuberin, which are encoded by the genes TSC1 and TSC2 that are mutated in the tuberous sclerosis complex disease. Recently, several laboratories have independently reported a major breakthrough in this field. Together, these genetic, biochemical and cell-biological studies have demonstrated that the tuberin-hamartin complex inhibits target of rapamycin (TOR) signaling by acting as a GTPase-activating protein for the Ras-related small G protein Rheb.

Homers are scaffolding proteins that bind G protein-coupled receptors (GPCRs), inositol 1,4,5-triphosphate ([IP.sub.3]) receptors ([IP.sub.3]Rs), ryanodine receptors, and TRP channels. However, their role in [Ca.sub.2+] signaling in vivo is not known. Characterization of [Ca.sub.2+] signaling in pancreatic acinar cells from Homer[2.sup.-/-] and Homer[3.sup.-/-] mice showed that Homer 3 has no discernible role in [Ca.sup.2+] signaling in these cells. In contrast, we found that Homer 2 tunes intensity of [Ca.sup.2+] signaling by GPCRs to regulate the frequency of [Ca.sup.2+]; oscillations. Thus, deletion of Homer 2 increased stimulus intensity by increasing the potency for agonists acting on various GPCRs to activate PLC[beta] and evoke [Ca.sup.2+] release and oscillations. This was not due to aberrant localization of I[P.sub.3]Rs in cellular microdomains or I[P.sub.3]R channel activity. Rather, deletion of Homer 2 reduced the effectiveness of exogenous regulators of G proteins signaling proteins (RGS) to inhibit [Ca.sup.2+] signaling in vivo. Moreover, Homer 2 preferentially bound to PLC[beta] in pancreatic acini and brain extracts and stimulated GAP activity of RGS4 and of PLC[beta] in an in vitro reconstitution system, with minimal effect on PLC3-mediated PI[P.sub.2] hydrolysis. These findings describe a novel, unexpected function of Homer proteins, demonstrate that RGS proteins and PLC[beta] GAP activities are regulated functions, and provide a molecular mechanism for tuning signal intensity generated by GPCRs and, thus, the characteristics of [Ca.sup.2+]; oscillations.

The sense of smell is arguably our most primal faculty and also the least understood. Even our own olfactorily impaired species is capable of detecting [approximately equal to] 10,000 distinct scents [Buck, L. & Axel, R. (1991) Cell 65, 175-1871. To achieve that amazing diversity, mammals have [approximately equal to] 1,000 olfactory genes, which accounts for [approximately equal to] 3% of their entire genome [Mombaerts, P. (1999) Science 286, 707-711]. The olfactory receptors (ORs) are believed to be seven-helix transmembrane proteins, with an odorant-binding site on the periplasmic domain and a G protein-binding site on the cytoplasmic domain. Odorants first bind to an OR, which then undergoes some structural change that triggers the G protein activation and the following cascade of events leading to nerve cell activity. The structural details of ORs, however, remain to be determined. In this paper, we will describe a hypothesis in which metal ions play an important role for odorant recognition. We analyze the predicted structure and consensus sequence of the ORs and propose a metal-binding site in the loop between fourth and fifth helix (4-5 loop). We have prepared synthetically a pentapeptide that contains this putative binding site and find that it not only has high affinity for binding Cu(II) and Zn(II) ions, but that it also undergoes a dramatic transition to an [alpha]-helical structure upon metal ion binding. Based on these observations, we propose a 'shuttlecock' mechanism for the possible structural change in ORs upon odorant binding. This mechanism involves membrane penetration of the 4-5 loop after residue charge neutralization by metal ion binding. olfaction | G protein-coupled receptors | transmembrane protein

Endothelin-A (ETA) is a G-protein-coupled receptor expressed in the neural crest-derived mesenchyme of the pharyngeal arches during craniofacial development. Targeted deletion of the [ET.sub.A] receptor or its ligand endothelin-1 (ET-1) causes cleft palate and hypoplasia of the mandible, otic cup, and tympanic ring. Previously we showed that G[[alpha].sub.q]/G[[alpha].sub.11]-null mice die around E 11.0, whereas G[[alpha].sub.q.sup.(-/-)] G[[alpha].sub.11.sup.(+/-)] mice survive to birth with hypomorphic phenotypes similar to, but less severe than, ETA or ET-1-null mice. To determine whether ET-1 signaling is transduced by G[[alpha].sub.q]/G[[alpha].sub.11] proteins, we examined the expression patterns of several ET-1 dependent and independent transcription factors in G[alpha].sub.q]/G[[alpha].sub.11]-deficient embryos. Expression of genes encoding the ET-1-dependent transcription factors D1x3, D1x6, dHAND, and eHAND was specifically downregulated in the pharyngeal arches of G[[alpha].sub.q]/G[alpha].sub.11]-deficient mice. In contrast, pharyngeal arch expression of the homeobox gene Msx1, which is not regulated by ET-1 signaling, was maintained in these embryos. We conclude that the G[[alpha].sub.q] and G[[alpha].sub.11] proteins serve as the intracellular mediators of ET-1 signaling in the pharyngeal arch mesenchyme. Keywords: G-protein; Neural crest; Pharyngeal arches; Endothelin; G[[alpha].sub.q]/G[[alpha].sub.11]; D1x3; D1x6; dHAND/HAND2; eHAND/HAND1; Craniofacial development

We recently identified an angiogenic mitogen, endocrine-gland-derived vascular endothelial growth factor (EG-VEGF), with selective activity for endothelial cells of endocrine tissues. Here we describe the characterization of a highly related molecule, Bv8, also known as prokineticin-2. Human Bv8 shares 60% identity and 75% similarity with EG-VEGF. The human and mouse By8 genes share a common structure. Like EG-VEGF, Bv8 is able to induce proliferation, survival and migration of adrenal cortical capillary endothelial cells. Bv8 gene expression is induced by hypoxic stress. Bv8 expression occurs predominantly in the testis and is largely restricted to primary spermatocytes. Adenoviral delivery of Bv8 or EG-VEGF to the mouse testis resulted in a potent angiogenic response. We have localized the expression of the Bv8/EG-VEGF receptors within the testis to vascular endothelial cells. The testis exhibits relatively high turnover of endothelial cells. Therefore, Bv8 and EG-VEGF, along with other factors such as VEGF-A, may maintain the integrity and also regulate proliferation of the blood vessels in the testis. mitogen | survival | migration | G protein-coupled receptor | prokineticin

In the G protein-coupled receptor rhodopsin, the conserved NPxxY[(x).sub.5,6]F motif connects the transmembrane helix VII and the cytoplasmic helix 8. The less geometrically constrained retinal analogue 9-demethyl-retinal prevents efficient transformation of rhodopsin to signaling metarhodopsin (Meta) II after retinal photoisomerization. Here, we demonstrate that Ala replacement mutations within the NPxxY[(x).sub.5,6]F domain, which eliminate an interaction between aromatic residues Y306 and F313, allow formation of Meta II despite the presence of 9-demethyl-retinal. Also a disulfide bond linking residues 306 and 313 in the 9-demethylretinal-reconstituted mutant Y306C/F313C/C316S prevented Meta II formation, whereas the reduced form of the mutant readily transformed to Meta II after illumination. These observations suggest that the interaction between residues 306 and 313 is disrupted during the Meta I/Meta II transition. However, this enhancement in Meta II formation is not reflected in the G protein activation, which is dramatically reduced for these mutants, suggesting that changes in the Y306-F313 interaction also lead to a proper realigning of helix 8 after photoisomerization. The E134Q mutation, located in the second conserved motif, D(E)RY, rescues activity in 9-demethyl-retinal-reconstituted mutants to different degrees, depending on the position of the Ala replacement in the NPxxY[(x).sub.5,6]F motif, thus revealing distinct roles for the NP and Y[(x).sub.5,6]F portions. Our studies underscore the importance of the NPxxY[(x).sub.5,6]F and D(E)RY motifs in providing structural constraints in rhodopsin that rearrange in response to photoisomerization during formation of the G protein-activating Meta II. The dual control of the structural rearrangements secures reliable transformation of quiescent rhodopsin to activating Meta II. GPCR | NPxxY motif

Little is known about the mechanisms converting psychosocial stress into cellular dysfunction. Various genes, up-regulated in atherosclerosis but also by psychosocial stress, are controlled by the transcription factor nuclear factor [kappa]B (NF-[kappa]B). Therefore, NF-[kappa]B is a good candidate to convert psychosocial stress into cellular activation. Volunteers were subjected to a brief laboratory stress test and NF-[kappa]B activity was determined in peripheral blood mononuclear cells (PBMC), as a window into the body and because PBMC play a role in diseases such as atherosclerosis. In 17 of 19 volunteers, NF-[kappa]B was rapidly induced during stress exposure, in parallel with elevated levels of catecholamines and cortisol, and returned to basal levels within 60 min. To model this response, mice transgenic for a strictly NF-[kappa]B-Controlled [beta]-globin transgene were stressed by immobilization. Immobilization resulted in increased [beta]-globin expression, which could be reduced in the presence of the [alpha]1-adrenergic inhibitor prazosin. To define the role of adrenergic stimulation in the up-regulation of NF-[kappa]B, THP-1 cells were induced with physiological amounts of catecholamines for 10 min. Only noradrenaline resulted in a dose- and time-dependent induction of NF-[kappa]B and NF-[kappa]B-dependent gene expression, which depended on pertussis-toxin-sensitive G protein-mediated phosphophatidylinositol 3-kinase, Ras/Raf, and mitogen-activated protein kinase activation. Induction was reduced by [[alpha].sub.1]- and [beta]-adrenergic inhibitors. Thus, noradrenaline-dependent adrenergic stimulation results in activation of NF-[kappa]B in vitro and in vivo. Activation of NF-[kappa]B represents a downstream effector for the neuroendocrine response to stressful psychosocial events and links changes in the activity of the neuroendocrine axis to the cellular response.

Research has been conducted on the arginine substitutions in the DRY motif (aspartic acid, arginine and tyrosine). The effect of these substitutions in alpha (sub)1B -adrenergic receptors and angiotensin II type 1A receptors on their localization, signaling and desensitization has been studied and the results are presented.

Aptamers, RNA sequences that bind to target ligands, are typically isolated by in vitro selection from RNA libraries containing completely random sequences. To see whether higher-affinity aptamers can be isolated from partially structured RNA libraries, we selected for aptamers that bind GTP, starting from a mixture of fully random and partially structured libraries. Because stem-loops are common motifs in previously characterized aptamers, we designed the partially structured library to contain a centrally located stable stem-loop. We used an off-rate selection protocol designed to maximize the enrichment of high-affinity aptamers. The selection produced a surprisingly large number of distinct sequence motifs and secondary structures, including seven different aptamers with [K.sub.d]s ranging from 500 to 25 nanomolar. The engineered stem-loop was present in the three highest affinity aptamers, and in 12 of 13 independent isolates with a single consensus sequence, suggesting that its inclusion increased the abundance of high-affinity aptamers in the starting pool.

Research has been conducted on the heterotrimeric G-protein alpha-subunit family. The gene encoding these G-protein alpha-subunits in homobasidiomycete Schizophyllum commune have been investigated and the active mutants have been generated via site-directed mutagenesis and used in examining the gene functions.

The [G.sub.12] subfamily of heterotrimeric G-proteins consists of two members, [G.sub.12] and [G.sub.13]. Gene-targeting studies have revealed a role for [G.sub.13] in blood vessel development. Mice lacking the [alpha] subunit of [G.sub.13] die around embryonic day 10 as the result of an angiogenic defect. On the other hand, the physiological role of [G.sub.12] is still unclear. To address this issue, we generated G[[alpha].sub.12]-deficient mice. In contrast to the G[[alpha].sub.13]-deficient mice, G[[alpha].sub.12]-deficient mice are viable, fertile, and do not show apparent abnormalities. However, G[[alpha].sub.12] does not seem to be entirely redundant, because in the offspring generated from G[[alpha].sub.12] [+ or -] G[[alpha].sub.13] [+ or -] intercrosses, at least one intact G[[alpha].sub.12] allele is required for the survival of animals with only one G[[alpha].sub.13] allele. In addition, G[[alpha].sub.12] and G[[alpha].sub.13] showed a difference in mediating cell migratory response to lysophosphatidic acid in embryonic fibroblast cells. Furthermore, mice lacking both G[[alpha].sub.12] and G[[alpha].sub.q] die in utero at about embryonic day 13. These data indicate that the G[[alpha].sub.12]-mediated signaling pathway functionally interacts not only with the G[[alpha].sub.13]- but also with the G[[alpha].sub.q/11]-mediated signaling systems.

Research has been conducted on the transmembrane segment Phe (super)282 residue conserved in beta (sub)2 -adrenergic receptors. The evaluation of this residue has been carried out via its mutation to glycine aspargine, alanine and leucine and the role of Phe (super)282 in G protein activation has been investigated.

Research has been conducted on the SWS2 class of visual pigments which are G-protein-coupled receptors. The evolution of these visual pigments in the cottoid fish species exposed to photic environments and the spectral tuning within the SWS2 group of visual pigments have been investigated and discussed.

Rhodopsin is the G protein-coupled receptor that is activated by light and initiates the transduction cascade leading to night (rod) vision. Naturally occurring pathogenic rhodopsin (RHO) mutations have been previously identified only in humans and are a common cause of dominantly inherited blindness from retinal degeneration. We identified English Mastiff dogs with a naturally occurring dominant retinal degeneration and determined the cause to be a point mutation in the RHO gene (Thr4Arg). Dogs with this mutant allele manifest a retinal phenotype that closely mimics that in humans with RHO mutations. The phenotypic features shared by dog and man include a dramatically slowed time course of recovery of rod photoreceptor function after light exposure and a distinctive topographic pattern to the retinal degeneration. The canine disease offers opportunities to explore the basis of prolonged photoreceptor recovery after light in RHO mutations and determine whether there are links between the dysfunction and apoptotic retinal cell death. The RHO mutant dog also becomes the large animal needed for preclinical trials of therapies for a major subset of human retinopathies.

Research has been conducted on the plant microtubules implicated into cell division and cell expansion. Results indicate that Arabidopsis PILZ group genes encode mammalian tubulin-folding cofactors and the associated small G-protein.

Many G protein-coupled receptors (GPCRs) have recently been shown to dimerize, and it was suggested that dimerization may be a prerequisite for G protein coupling. [gamma]-aminobutyric acid type B (GAB[A.sup.B]) receptors (GPCRs for GABA, a major inhibitory neurotransmitter in the brain) are obligate heterodimers of homologous GB1 and GB2 subunits, neither of which is functional on its own. This feature of GAB[A.sub.B] receptors allowed us to examine which of the eight intracellular segments of the heterodimeric receptor were important for G protein activation. Replacing any of the three intracellular loops of GB2 with their GB1 counterparts resulted in nonfunctional receptors. The deletion of the complete GB2 C terminus significantly attenuated the receptor function; however, the proximal 36 residues were sufficient for reconstitution of wild type-like receptor activity. In contrast, the GB1 C terminus could be deleted and GB1 intracellular loops replaced with their GB2 or mGluR1 equivalents without affecting the receptor function. In addition, a large portion of the GB1 i2 loop could be replaced with a random coli peptide without any functional consequences. Thus, GB2 intracellular segments are solely responsible for specific coupling of GAB[A.sub.B] receptors to their physiologic effectors, [G.sub.i] and G protein-activated [K.sup.+] channels. These findings strongly support a model in which a single GPCR monomer is sufficient for all of the specific G protein contacts.

Research has been conducted on the mutations within the X (sub)1 BBX (sub)2 X (sub)3 B motif that lead to the constitutive activation of the G protein-coupled receptors. The mutation of the Thr6.34(279) to Lys and Asp at the X (sub)3 locus of the rat mu opioid receptor has been carried out and the binding and signaling properties of the mutants have been examined.

A sordarin-specificity region in eukaryotic elongation factor 2 (eEF2) has been identified in study of species-specific inhibition of fungal protein synthesis by sordarin. The cEF2s from various sordarin-sensitive and -insensitive species were cloned and characterized. Amino acid residues at three positions in fungal eEF2 were mainly responsible the differences seen in sordarin's spectrum. The corresponding residues in mammalian eEF2 are adequate to give a sordarin-insensitive phenotype to S. cerevisiae eEF2.

Results show the RHO1 protein of Cryptococcus neoformans can complement Saccharomyces cerevisiae deficient in the synthesis of this protein. Data also suggest that a mutation in the gene of the bacterium leads to different growth characteristics than of those yeast mutants.

Regulators of G protein signaling (RGS proteins) have been studied in Caenorhabditis elegans. Three of the 13 RGS genes analyzed were found to influence egg-laying behavior and two seem to redundantly change signaling to bring on appropriate behavioral responses to feeding.

Research indicates that Obg is a GTP binding protein and participates in the stress activation of sigma factor B in Bacillus subtilis. Data further suggest that Obg and sigma factor B functions are possibly mediated by the ribosomes.

The general stress response sigma factor, sigmaB, of Bacillus subtilis is activated when intracellular ATP levels fall or the bacterium is exposed to environmental stress. By employing the yeast dihybrid selection to identify B. subtilis proteins that could interact with Rsb proteins and act as mediators of stress signaling, the G protein Obg was isolated as an interactor with several of these regulators. B. subtilis depleted of Obg no longer activated sigmaB in response to environmental stress.

The ability of the conserved aspartate residue D98 and the arginine residue R309 in binding agonist and catalyzing exchange of guanine nucleotides has been studied by expressing site-directed gastrin-releasing peptide receptor (GRP-R) mutants in fibroblasts. The GRP-R is a G protein-coupled receptor belonging to the bombesin receptor subfamily. The D98 is located at the extracellular boundary of transmembrane domain II and the R309 is near the extracellular boundary of transmembrane domain VII. Results indicate that agonist binding affinity is reduced 24-fold at D98 and 56-fold at R309. Agonist binding in double-swap mutations was only six times lower than in wild-type GRP-R.

Several regulators of G protein signaling (RGS) proteins contain a G protein [Gamma]-subunit-like (GGL) domain, which, as we have shown, binds to [G.sub.[Beta]5] subunits. Here, we extend our original findings by describing another GGL-domain-containing RGS, human RGS6. When RGS6 is coexpressed with different [G.sub.[Beta]] subunits, only RGS6 and [G.sub.[Beta]5] interact. The expression of mRNA for RGS6 and [G.sub.[Beta]5] in human tissues overlaps. Predictions of [Alpha]-helical and coiled-coil character within GGL domains, coupled with measurements of [G.sub.[Beta]] binding by GGL domain mutants, support the contention that [G.sub.[Gamma]]-like regions within RGS proteins interact with [G.sub.[Beta]5] subunits in a fashion comparable to conventional [G.sub.[Beta]]/[G.sub.[Gamma]] pairings. Mutation of the highly conserved Phe-61 residue of [G.sub.[Gamma]2] to tryptophan, the residue present in all GGL domains, increases the stability of the [G.sub.[Beta]5]/[G.sub.[Gamma]2] heterodimer, highlighting the importance of this residue to GGL/[G.sub.[Beta]5] association.