Receptor activity modifying proteins (RAMPs) interact with the VPAC2 receptor and CRF1 receptors and modulate their function.

BACKGROUND AND PURPOSE: Although it is established that the receptor activity modifying proteins (RAMPs) can interact with a number of GPCRs, little is known about the consequences of these interactions. Here the interaction of RAMPs with the glucagon-like peptide 1 receptor (GLP-1 receptor), the human vasoactive intestinal polypeptide/pituitary AC-activating peptide 2 receptor (VPAC(2)) and the type 1 corticotrophin releasing factor receptor (CRF(1)) has been examined. EXPERIMENTAL APPROACH: GPCRs were co-transfected with RAMPs in HEK 293S and CHO-K1 cells. Cell surface expression of RAMPs and GPCRs was examined by ELISA. Where there was evidence for interactions, agonist-stimulated cAMP production, Ca(2+) mobilization and GTPγS binding to G(s), G(i), G(12) and G(q) were examined. The ability of CRF to stimulate adrenal corticotrophic hormone release in Ramp2(+/-) mice was assessed. KEY RESULTS: The GLP-1 receptor failed to enhance the cell surface expression of any RAMP. VPAC(2) enhanced the cell surface expression of all three RAMPs. CRF(1) enhanced the cell surface expression of RAMP2; the cell surface expression of CRF(1) was also increased. There was no effect on agonist-stimulated cAMP production. However, there was enhanced G-protein coupling in a receptor and agonist-dependent manner. The CRF(1) : RAMP2 complex resulted in enhanced elevation of intracellular calcium to CRF and urocortin 1 but not sauvagine. In Ramp2(+/-) mice, there was a loss of responsiveness to CRF. CONCLUSIONS AND IMPLICATIONS: The VPAC(2) and CRF(1) receptors interact with RAMPs. This modulates G-protein coupling in an agonist-specific manner. For CRF(1), coupling to RAMP2 may be of physiological significance.

The orphan receptor GPR55 is a novel cannabinoid receptor.

BACKGROUND: The endocannabinoid system functions through two well characterized receptor systems, the CB1 and CB2 receptors. Work by a number of groups in recent years has provided evidence that the system is more complicated and additional receptor types should exist to explain ligand activity in a number of physiological processes. EXPERIMENTAL APPROACH: Cells transfected with the human cDNA for GPR55 were tested for their ability to bind and to mediate GTPgammaS binding by cannabinoid ligands. Using an antibody and peptide blocking approach, the nature of the G-protein coupling was determined and further demonstrated by measuring activity of downstream signalling pathways. KEY RESULTS: We demonstrate that GPR55 binds to and is activated by the cannabinoid ligand CP55940. In addition endocannabinoids including anandamide and virodhamine activate GTPgammaS binding via GPR55 with nM potencies. Ligands such as cannabidiol and abnormal cannabidiol which exhibit no CB1 or CB2 activity and are believed to function at a novel cannabinoid receptor, also showed activity at GPR55. GPR55 couples to Galpha13 and can mediate activation of rhoA, cdc42 and rac1. CONCLUSIONS: These data suggest that GPR55 is a novel cannabinoid receptor, and its ligand profile with respect to CB1 and CB2 described here will permit delineation of its physiological function(s).

Adenosine is not a direct GHSR agonist--artificial cross-talk between GHSR and adenosine receptor pathways.

AIM: To assess if adenosine is a direct growth hormone secretagogue receptor (GHSR) agonist by investigating the mechanism behind adenosine induced calcium release in human embryonic kidney 293s (HEK) cells expressing GHSR. METHODS: Calcium mobilization, cyclic adenosine monophosphate (cAMP) and IP(3) experiments were performed using HEK cells stably expressing GHSR and/or adenosine A(2B) receptor (A(2B)R). RESULTS: Adenosine has been widely reported as a GHSR agonist. In our hands, adenosine and forskolin stimulated calcium release from IP(3) controlled stores in HEK-GHSR cells but not in non-transfected HEK cells. This release was not accompanied by increased IP(3) levels. The calcium release was both cholera toxin and U73122 sensitive, indicating the involvement of both Galpha(s)/adenylyl cyclase and Galpha(q/11)/phospholipase C pathways. Importantly, the GHSR inverse agonist [D-Arg(1) D-Phe(5) D-Trp(7,9) Leu(11)]-Substance P (SP-analogue) blocked the adenosine stimulated calcium release, demonstrating that GHSR is involved. Assessment of the GHSR-dependent calcium release using adenosine receptor agonists and antagonists resulted in a rank order of potencies resembling the profile of A(2B)R. A(2B)R over-expression in HEK-GHSR cells enhanced potency and efficacy of the adenosine induced calcium release without increasing IP(3) production. Moreover, A(2B)R over-expression in HEK cells potentiated NECA-induced cAMP production. However, GHSR expression had no effect on intracellular cAMP production. CONCLUSION: In HEK-GHSR cells adenosine activates endogenously expressed A(2B)R resulting in calcium mobilization. We hypothesize that the responsible mechanism is cAMP-dependent sensitization of IP(3) receptors for the high basal level of IP(3) caused by GHSR constitutive activity. Altogether, our results demonstrate that adenosine is not a direct GHSR agonist.

Biochemistry of transmembrane signaling mediated by trimeric G proteins.

Many extracellular signals are at the cell surface received by specific receptors, which upon activation transduce information to the appropriate cellular effector molecules via trimeric G proteins. The G protein-mediated cascades ultimately lead to the highly refined regulation of systems such as sensory perception, cell growth, and hormonal regulation. Transmembrane signaling may be seriously deranged in various pathophysiological conditions. Over the last two decades the major experimental effort of our group has been devoted to better understanding the molecular mechanisms underlying transmembrane signaling regulated by G proteins and to the closely related process of desensitization of hormone response. This review provides general information about the basic principles of G protein-regulated transmembrane signaling as well as about our contribution to the current progress in the field.

Analysis of the C-terminal tail of the rat thyrotropin-releasing hormone receptor-1 in interactions and cointernalization with beta-arrestin 1-green fluorescent protein.

Coexpression of the rat thyrotropin releasing hormone receptor-1 with beta-arrestin 1-green fluorescent protein (GFP) in human embryonic kidney 293 cells results in agonist-dependent translocation of the arrestin to the plasma membrane followed by its cointernalization with the receptor. Truncations of the receptor C-terminal tail from 93 to 50 amino acids did not alter this. Truncations to fewer than 47 amino acids prevented such interactions and inhibited but did not fully eliminate agonist-induced internalization of the receptor. Deletion and site-directed mutants of the C-terminal tail indicated that separate elimination of a potential casein kinase II phosphorylation site or clathrin/clathrin adapter motifs was insufficient to prevent either internalization of the receptor or its cointernalization with beta-arrestin 1-GFP. Alteration of sites of acylation reduced internalization and prevented interactions with beta-arrestin 1-GFP. Combinations of these mutants resulted in lack of interaction with beta-arrestin 1-GFP and a 10-fold reduction in internalization of the receptor. Despite this, the receptor construct that lacked the three protein sequence motifs was fully functional. These studies map sites that contribute the interactions of the thyrotropin releasing hormone receptor-1 C-terminal tail required for effective contacts with beta-arrestin 1-GFP and indicate key roles for these interactions in agonist-induced internalization of the receptor.

Kinetic analysis of the internalization and recycling of [3H]TRH and C-terminal truncations of the long isoform of the rat thyrotropin-releasing hormone receptor-1.

The C-terminal tail of the long splice variant of the rat thyrotropin-releasing hormone (TRH) receptor-1 (TRHR-1L) comprises around 93 amino acids. A series of C-terminal truncations was constructed and expressed transiently in HEK-293 cells. The extent of steady-state internalization of these in response to [(3)H]TRH was dependent upon the degree of truncation. Little effect was produced by deletion of the C-terminal to 50 amino acids, although there was a substantial decrease in the extent of internalization by deletion to 45-46 amino acids. The rate of internalization of TRHR-1L in response to ligand was substantially decreased by the acid-wash procedures often used in the analysis of cellular distribution of receptors with peptide ligands, and thus an alternative procedure using a Mes-containing buffer was employed in the present study. Apart from a truncation anticipated to eliminate post-translational acylation of the re-ceptor, which altered both the association and dissociation rates of [(3)H]TRH, the kinetics of ligand binding were unaffected by C-terminal truncation. Equally, the rate of recycling to the plasma membrane of internalized receptors was unaffected by C-terminal truncation. Although the extent of internalization of the full-length receptor was impaired by pre-exposure of cells to TRH, this was not true of C-terminal truncation mutants, which displayed limited steady-state internalization ratios. A mutant with a substantial C-terminal deletion also displayed decreased functional desensitization compared with the full-length receptor.

Functional analysis of a human A(1) adenosine receptor/green fluorescent protein/G(i1)alpha fusion protein following stable expression in CHO cells.

Fusion proteins between the human A(1) adenosine receptor and the pertussis toxin resistant (Cys351Gly) mutant of the G-protein alpha subunit G(i1)alpha (A1/Gi), and between the human A(1) adenosine receptor, the Aequorea victoria green fluorescent protein (GFP) and Cys351Gly G(i1)alpha (A1/GFP/Gi), were expressed in CHO cells. The agonist NECA caused a stimulation of [(35)S]GTPgammaS binding at both fusion proteins with similar concentration dependence as at the native receptor. However in the presence of pertussis toxin NECA stimulation of [(35)S]GTPgammaS binding was only seen at the A1/GFP/Gi fusion protein. The regulation of the adenylyl cyclase and MAP kinase effector systems by both fusion proteins was attenuated following pertussis toxin treatment. These studies demonstrate for the first time the characterisation of a fusion protein between a G-protein coupled receptor, GFP and a G-protein alpha subunit.

Overexpression of the G protein G11alpha prevents desensitization of Ca2+ response to thyrotropin-releasing hormone.

Doubly transfected human embryonal kidney cells (clone E2M11 of the HEK 293 cell line) expressing both thyrotropin-releasing hormone (TRH) receptors and G11alpha protein in high amounts were used to analyze the desensitization phenomenon of the Ca2+-mobilizing pathway. Quite unexpectedly, we did not observe any significant desensitization of the [Ca2+]i response to TRH in these cells after repeated or prolonged incubation with the hormone (up to 5 h). Under the same conditions, the TRH-induced [Ca2+]i response was completely desensitized in the parent cell line (293-E2 cels) expressing TRH receptors alone. In both cell lines, inositol phosphate response was desensitized after TRH exposure, although basal levels of inositol phospates in TRH-pretreated cells were much higher than in "naive" TRH-unexposed cells. These data suggest a significant role of the G protein G11alpha in desensitization of the Ca2+-mobilizing pathway occuring after repeated or long-term exposure of target cells to TRH-receptor agonists.

Visualization of distinct patterns of subcellular redistribution of the thyrotropin-releasing hormone receptor-1 and gqalpha /G11alpha induced by agonist stimulation.

The rat thyrotropin-releasing hormone receptor-1 (TRHR-1) was modified by the addition of green fluorescent protein (GFP) and expressed stably in HEK293 cells. Extensive overlap of plasma membrane distribution of autofluorescent TRHR-1-GFP with that of the phosphoinositidase C-linked G-proteins Gqalpha/G11alpha, identified by indirect immunofluorescence, was monitored concurrently. Addition of thyrotropin-releasing hormone resulted in rapid separation of TRHR-1-GFP and Gqalpha/G11alpha signals as the receptor was internalized. This situation persisted for more than an hour. At longer time periods a fraction of the cellular Gqalpha/G11alpha was also internalized, although much of the Gqalpha/G11alpha immunoreactivity remained associated with the plasma membrane. Parallel experiments, in which the cellular distribution of TRHR-1-GFP and Gqalpha/G11alpha immunoreactivity were monitored in sucrose-gradient fractions following cell disruption, also demonstrated a rapid, agonist-induced movement of TRHR-1-GFP away from the plasma membrane to low-density vesicular fractions. At later time points, a fraction of the cellular Gqalpha/G11alpha immunoreactivity was also redistributed to overlapping, but non-identical, low-density-vesicle-containing fractions. Pretreatment of the cells with cytochalasin D or nocodazole prevented agonist-induced redistribution of G-protein but not TRHR-1-GFP, further indicating resolution of the mechanics of these two processes. The combination of a GFP-modified receptor and immunostaining of the G-proteins activated by that receptor allows, for the first time, concurrent analysis of the varying dynamics and bases of internalization and redistribution of two elements of the same signal-transduction cascade.

Real time visualization of agonist-mediated redistribution and internalization of a green fluorescent protein-tagged form of the thyrotropin-releasing hormone receptor.

The long isoform of the rat thyrotropin-releasing hormone receptor (TRHR) was modified by the addition of a vesicular stomatitis virus (VSV) epitope tag and green fluorescent protein (GFP). VSV-TRHR-GFP bound TRH with affinity similar to that of the unmodified receptor and stimulated [3H]inositol phosphate production. A clone stably expressing VSV-TRHR-GFP at some 120,000 copies/cell was selected to visualize this receptor during cellular exposure to TRH. Internalization was detected within 3-5 min after treatment with 1 x 10(-7) M TRH, with dramatic reductions in plasma membrane localization achieved within 10-15 min. The TRHR antagonist/inverse agonist chlordiazepoxide competitively inhibited internalization. Hyperosmotic sucrose inhibited internalization of VSV-TRHR-GFP, measured both by intact cell [3H]TRH binding studies and by confocal microscopy. Now TRH caused a redistribution of VSV-TRHR-GFP to highly punctate but plasma membrane-delineated foci. Pretreatment with the microtubule-disrupting agent nocodazole allowed internalization of the VSV-TRHR-GFP construct but only into vesicles that remained in close apposition to the plasma membrane. Covisualization of VSV-TRHR-GFP and Texas Red transferrin initially indicated entirely separate localizations. After exposure to TRH substantial amounts of VSV-TRHR-GFP were present in vesicles overlapping those containing Texas Red transferrin. Such results demonstrate the G protein-coupling capacity and provide real time visualization of the processes of internalization of a TRH-receptor-GFP construct in response to agonist.

Agonist-induced internalization of the G protein G11alpha and thyrotropin-releasing hormone receptors proceed on different time scales.

Using a combination of confocal immunofluorescence microscopy and subcellular fractionation, we demonstrate for the first time active internalization, trafficking, and down-regulation of a G protein alpha subunit subsequent to agonist occupation of a receptor. This proceeds on a much slower time scale than internalization of the corresponding receptor. In intact E2M11 HEK293 cells that express high levels of murine G11alpha and the rat thyrotropin-releasing hormone (TRH) receptor, the immunofluorescence signal of G11alpha was restricted almost exclusively to the plasma membrane. Exposure to TRH (10 microM) resulted first in partial relocation of G11alpha to discrete, segregated patches within the plasma membrane (10-60 min). Further exposure to TRH caused internalization of G11alpha to discrete, punctate, intracellular bodies (2-4 h) and subsequently to a virtually complete loss of G11alpha from plasma membranes and the cells (8-16 h). Short-term treatment with TRH followed by wash-out of the ligand allowed G11alpha immunofluorescence to be restored to the plasma membrane within 12 h. In subcellular membrane fractions, G11alpha was centered on plasma membranes, and this was not altered by up to 1-2 h of incubation with TRH. Further exposure to TRH (2-4 h) resulted in transfer of a significant portion of G11alpha to light-vesicular and cytosol fractions. At longer time intervals (4-16 h), an overall decrease in G11alpha content was observed.

Isolation and characterization of cytosolic malate dehydrogenase from Trichomonas vaginalis.

Malate dehydrogenase (EC 1.1.1.37.) (MDH) was purified to apparent homogeneity from the cytosolic fraction of the protozoan Trichomonas vaginalis Donné. The four step purification included ion-exchange chromatography (DEAE-Sephacel and Q-Sepharose, elution with NaCl) and affinity chromatography (Reactive Red Agarose, elution with NADH and NaCl). The enzyme was purified about 132-fold (30.6% yield) to a specific activity of 352 units mg-1. The Km values determined at pH 7.8 (pH optimum from 7.5 to 8.3) for oxaloacetate and NADH were 16.2 microM and 10.6 microM, respectively. The MDH activity was inhibited by the substrate, decreasing to 50% at about 1 microM concentration of oxaloacetate. The reverse reaction from malate to oxaloacetate showed a pH optimum around pH 9.5. The Km for malate and NAD+ (determined at pH 7.8) were 1220 microM and 69.9 microM, respectively. SDS-PAGE analysis of the purified MDH revealed a single band with an apparent size of 34.5 kDa. The native molecular weight was estimated by HPLC gel filtration to be 60 kDa, which indicates that the T. vaginalis MDH exists as a dimer.

Iron-ascorbate cleavable malic enzyme from hydrogenosomes of Trichomonas vaginalis: purification and characterization.

Two isoforms of NAD(P)(+)-dependent malic enzyme (EC 1.1.1.39) were isolated from hydrogenosomes of Trichomonas vaginalis. A positively charged isoform at pH 7 was obtained in a single purification step using cation-exchange chromatography. The second isoform, negatively charged at pH 7.5, was partially purified using a combination of anion-exchange and affinity chromatography. Both isoforms displayed similar physical and kinetic properties. Molecular weight determination of the native enzyme suggested a homotetrameric arrangement of the 60 kDa subunits. The enzyme utilized NAD+ (Km, 6-6.3 microM) preferentially to NADP+ (Km, 125-145 microM). The NAD(+)-dependent activity showed a broad pH optimum with maximum under alkaline conditions (pH 9) likely to be present inside hydrogenosomes. Immunocytochemical studies using a polyclonal rabbit antibody raised against purified T. vaginalis malic enzyme proved hydrogenosomal localization of the enzyme. Subfractionation of hydrogenosomes suggested an association of the malic enzyme with the hydrogenosomal membranes. The 60 kDa malic enzyme subunit was highly sensitive to non-enzymatic cleavage by an iron-ascorbate system resulting in two enzymatically inactive fragments of about 31 kDa. Microsequencing of the fragments revealed that the 60 kDa subunit was cleaved at the metal-binding site between Asp279-Ile280. The enzyme inactivation was inhibited by an excess of manganese. Iron-dependent posttranslational modification might contribute to the regulation of malic enzyme activity in vivo.

Cloning and characterization of the NAD-linked glycerol-3-phosphate dehydrogenases of Trypanosoma brucei brucei and Leishmania mexicana mexicana and expression of the trypanosome enzyme in Escherichia coli.

A polyclonal antiserum raised against the purified glycosomal glycerol-3-phosphate dehydrogenase of Trypanosoma brucei brucei has been used to identify the corresponding cDNA clone in a T.b. brucei expression library. This cDNA was subsequently used to obtain genomic clones containing glycerol-3-phosphate dehydrogenase genes. Two tandemly arranged genes were detected in these clones. Characterization of one of the genes showed that it codes for a polypeptide of 353 amino acids, with a molecular mass of 37,651 Da and a calculated net charge of +8. Using the T.b. brucei gene as a probe, a corresponding glycerol-3-phosphate dehydrogenase gene was also identified in a genomic library of Leishmania mexicana mexicana. The L.m. mexicana gene codes for a polypeptide of 365 amino acids, with a molecular mass of 39,140 Da and a calculated net charge of +8. The amino-acid sequences of both polypeptides are 63% identical and carry a type-1 peroxisomal targeting signal (PTS1) SKM and -SKL at their respective C-termini. Moreover, the L.m. mexicana polypeptide also carries a short N-terminal extension reminiscent of a mitochondrial transit sequence. Subcellular localisation analysis showed that in L.m. mexicana the glycerol-3-phosphate dehydrogenase activity co-fractionated both with mitochondria and with glycosomes. This is not the case in T. brucei, where the enzyme is predominantly glycosomal. The two trypanosomatid sequences resemble their prokaryotic homologues (32-36%) more than their eukaryotic counterparts (25-31%) and carry typical prokaryotic signatures. The possible reason for this prokaryotic nature of a trypanosomatid glycerol-3-phosphate dehydrogenase is discussed.