Previous studies have indicated that the G-protein-coupled secretin receptor is present as a homodimer, organized through symmetrical contacts in transmembrane domain IV, and that receptor dimerization is critical for high-potency signalling by secretin. However, whether all of the receptor exists in the dimeric form or if this is regulated is unclear. We used measures of quantal brightness of the secretin receptor tagged with monomeric enhanced green fluorescent protein (mEGFP) and spatial intensity distribution analysis to assess this. Calibration using cells expressing plasma membrane-anchored forms of mEGFP initially allowed us to demonstrate that the epidermal growth factor receptor is predominantly monomeric in the absence of ligand and while wild-type receptor was rapidly converted into a dimeric form by ligand, a mutated form of this receptor remained monomeric. Equivalent studies showed that, at moderate expression levels, the secretin receptor exists as a mixture of monomeric and dimeric forms, with little evidence of higher-order complexity. However, sodium butyrate-induced up-regulation of the receptor resulted in a shift from monomeric towards oligomeric organization. In contrast, a form of the secretin receptor containing a pair of mutations on the lipid-facing side of transmembrane domain IV was almost entirely monomeric. Down-regulation of the secretin receptor-interacting G-protein Gα s did not alter receptor organization, indicating that dimerization is defined specifically by direct protein–protein interactions between copies of the receptor polypeptide, while short-term treatment with secretin had no effect on organization of the wild-type receptor but increased the dimeric proportion of the mutated receptor variant.

Robo (Roundabout) receptors and their Slit polypeptide ligands are known to play key roles in neuronal development and have been implicated in both angiogenesis and cancer. Like the other family members, Robo1 is a large single transmembrane domain polypeptide containing a series of well-defined extracellular elements. However, the intracellular domain lacks structural definition and little is known about the quaternary structure of Robo receptors or how binding of a Slit might affect this. To address these questions combinations of both autofluorescent protein-based FRET imaging and time-resolved FRET were employed. Both approaches identified oligomeric organization of Robo1 that did not require the presence of the intracellular domain. SpIDA (spatial intensity distribution analysis) of eGFP-tagged forms of Robo1 indicated that for a C-terminally deleted version approximately two-thirds of the receptor was present as a dimer and one-third as a monomer. By contrast, full-length Robo1 was present almost exclusively as a dimer. In each case this was unaffected by the addition of Slit2, although parallel studies demonstrated the biological activity of Slit2 and its interaction with Robo1. Deletion of both the immunoglobulin and fibronectin type III extracellular repeats prevented dimer formation, with the immunoglobulin repeats providing the bulk of the protein–protein interaction affinity.

The literature on GPCR (G-protein-coupled receptor) homo-oligomerization encompasses conflicting views that range from interpretations that GPCRs must be monomeric, through comparatively newer proposals that they exist as dimers or higher-order oligomers, to suggestions that such quaternary structures are rather ephemeral or merely accidental and may serve no functional purpose. In the present study we use a novel method of FRET (Förster resonance energy transfer) spectrometry and controlled expression of energy donor-tagged species to show that M 3 Rs (muscarinic M 3 acetylcholine receptors) at the plasma membrane exist as stable dimeric complexes, a large fraction of which interact dynamically to form tetramers without the presence of trimers, pentamers, hexamers etc. That M 3 R dimeric units interact dynamically was also supported by co-immunoprecipitation of receptors synthesized at distinct times. On the basis of all these findings, we propose a conceptual framework that may reconcile the conflicting views on the quaternary structure of GPCRs.

It is unclear what proportion of a G-protein-coupled receptor is present in cells as dimers or oligomers. Saturation bioluminescence resonance energy transfer studies demonstrated the orexin OX 1 receptor to be present in such complexes. Forms of this receptor containing a minimal epitope tag, with the C-terminus linked to yellow fluorescent protein or modified at the N-terminus to incorporate a SNAP tag, migrated in SDS/PAGE gels as monomers, indicating a lack of covalent interactions. Solubilization with dodecylmaltoside, followed by Blue native-PAGE, indicated that the receptor constructs migrated predominantly as anticipated for dimeric species with evidence for further, higher-order, complexes, and this was true over a wide range of expression levels. Addition of SDS prior to separation by Blue native-PAGE resulted in much of the previously dimeric, and all of the higher-order, complexes being dissociated and now migrating at the size predicted for monomeric species. Expression of forms of the OX 1 receptor capable of generating enzyme complementation confirmed that solubilization itself did not result in interaction artefacts. Addition of the endogenous agonist orexin A enhanced the proportion of higher-order OX 1 receptor complexes, whereas selective OX 1 antagonists increased the proportion the OX 1 receptor migrating in Blue native-PAGE as a monomer. The antagonist effects were produced in a concentration-dependent manner, consistent with the affinity of the ligands for the receptor. Homogeneous time-resolved fluorescence resonance energy transfer studies using Tag-Lite™ reagents on cells expressing the SNAP-tagged OX 1 receptor identified cell-surface OX 1 homomers. Predominantly at low receptor expression levels, orexin A increased such fluorescence resonance energy transfer signals, also consistent with ligand-induced reorganization of the homomeric complex.

hESCs (human embryonic stem cells) have enormous potential for use in pharmaceutical development and therapeutics; however, to realize this potential, there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with EHNA [ erythro -9-(2-hydroxy-3-nonyl)adenine], an established inhibitor of ADA (adenosine deaminase) and cyclic nucleotide PDE2 (phosphodiesterase 2). hESCs maintained in feeder-free conditions with EHNA for more than ten passages showed no reduction in hESC-associated markers including NANOG, POU5F1 (POU domain class 5 transcription factor 1, also known as Oct-4) and SSEA4 (stage-specific embryonic antigen 4) compared with cells maintained in feeder-free conditions containing bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but, upon removing EHNA, hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure–activity relationship analysis found the differentiation-blocking properties of EHNA to reside in a pharmacophore comprising a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large-scale or cGMP-compliant processes.

The poorly characterized G-protein-coupled receptor GPR35 has been suggested as a potential exploratory target for the treatment of both metabolic disorders and hypertension. It has also been indicated to play an important role in immune modulation. A major impediment to validation of these concepts and further study of the role of this receptor has been a paucity of pharmacological tools that interact with GPR35. Using a receptor–β-arrestin-2 interaction assay with both human and rat orthologues of GPR35, we identified a number of compounds possessing agonist activity. These included the previously described ligand zaprinast. Although a number of active compounds, including cromolyn disodium and dicumarol, displayed similar potency at both orthologues of GPR35, a number of ligands, including pamoate and niflumic acid, had detectable activity only at human GPR35 whereas others, including zaprinast and luteolin, were markedly selective for the rat orthologue. Previous studies have demonstrated activation of G α13 by GPR35. A Saccharomyces cerevisiae -based assay employing a chimaeric Gpa1–G α13 G-protein confirmed that all of the compounds active at human GPR35 in the β-arrestin-2 interaction assay were also able to promote cell growth via G α13 . Each of these ligands also promoted binding of [ 35 S]GTP[S] (guanosine 5′-[γ-[ 35 S]thio]triphosphate) to an epitope-tagged form of G α13 in a GPR35-dependent manner. The ligands identified in these studies will be useful in interrogating the biological actions of GPR35, but appreciation of the species selectivity of ligands at this receptor will be vital to correctly attribute function.

The propensity of human embryonic stem cells to die upon enzymatic disaggregation or low-density plating is an obstacle to their isolation and routine use in drug discovery and basic research. Equally, the very low rate of establishment of implanted cells hinders cell therapy. In the present study we have developed a high-content assay for human embryonic stem cell survival and used this to screen a range of libraries of ‘lead-like’ small molecules and known bioactives. From this we identified 18 confirmed hits with four structural classes being represented by multiple compounds: a series of 5-(acyl/alkyl-amino)indazoles, compounds with a 4-(acylamino)pyridine core, simple N 6 , N 6 -dialkyladenines and compounds with a 5-(acylamino)indolinone core. In vitro kinase profiling indicated that the ROCK (Rho-associated kinase)/PRK2 (protein kinase C-related kinase 2) protein kinases are of pivotal importance for cell survival and identified previously unreported compound classes that inhibited this important biological activity. An evaluation using an extensive panel of protein kinases showed that six of our hit compounds exhibited better selectivity for ROCK inhibition than the routinely used commercially available ROCK inhibitor Y-27632. In this screen we also identified the K + -ATP channel opener pinacidil and show that it probably promotes cell survival, by ‘off-target’ inhibition of ROCK/PRK2. We have therefore identified novel pro-survival compounds of greater specificity, equivalent potency and reduced toxicity relative to the routinely employed ROCK inhibitor Y-27632.

Many G-protein-coupled receptors, including the α 1b -adrenoceptor, form homo-dimers or oligomers. Mutation of hydrophobic residues in transmembrane domains I and IV alters the organization of the α 1b -adrenoceptor oligomer, with transmembrane domain IV playing a critical role. These mutations also result in endoplasmic reticulum trapping of the receptor. Following stable expression of this α 1b -adrenoceptor mutant, cell surface delivery, receptor function and structural organization were recovered by treatment with a range of α 1b -adrenoceptor antagonists that acted at the level of the endoplasmic reticulum. This was accompanied by maturation of the mutant receptor to a terminally N-glycosylated form, and only this mature form was trafficked to the cell surface. Co-expression of the mutant receptor with an otherwise wild-type form of the α 1b -adrenoceptor that is unable to bind ligands resulted in this wild-type variant also being retained in the endoplasmic reticulum. Ligand-induced cell surface delivery of the mutant α 1b -adrenoceptor now allowed co-recovery to the plasma membrane of the ligand-binding-deficient mutant. These results demonstrate that interactions between α 1b -adrenoceptor monomers occur at an early stage in protein synthesis, that ligands of the α 1b -adrenoceptor can act as pharmacological chaperones to allow a structurally compromised form of the receptor to pass cellular quality control, that the mutated receptor is not inherently deficient in function and that an oligomeric assembly of ligand-binding-competent and -incompetent forms of the α 1b -adrenoceptor can be trafficked to the cell surface by binding of a ligand to only one component of the receptor oligomer.

FRET (fluorescence resonance energy transfer) and co-immunoprecipitation studies confirmed the capacity of β-arrestin 2 to self-associate. Amino acids potentially involved in direct protein–protein interaction were identified via combinations of spot-immobilized peptide arrays and mapping of surface exposure. Among potential key amino acids, Lys 285 , Arg 286 and Lys 295 are part of a continuous surface epitope located in the polar core between the N- and C-terminal domains. Introduction of K285A/R286A mutations into β-arrestin 2–eCFP (where eCFP is enhanced cyan fluorescent protein) and β-arrestin 2–eYFP (where eYFP is enhanced yellow fluorescent protein) constructs substantially reduced FRET, whereas introduction of a K295A mutation had a more limited effect. Neither of these mutants was able to promote β 2 -adrenoceptor-mediated phosphorylation of the ERK1/2 (extracellular-signal-regulated kinase 1/2) MAPKs (mitogen-activated protein kinases). Both β-arrestin 2 mutants displayed limited capacity to co-immunoprecipitate ERK1/2 and further spot-immobilized peptide arrays indicated each of Lys 285 , Arg 286 and particularly Lys 295 to be important for this interaction. Direct interactions between β-arrestin 2 and the β 2 -adrenoceptor were also compromised by both K285A/R286A and K295A mutations of β-arrestin 2. These were not non-specific effects linked to improper folding of β-arrestin 2 as limited proteolysis was unable to distinguish the K285A/R286A or K295A mutants from wild-type β-arrestin 2, and the interaction of β-arrestin 2 with JNK3 (c-Jun N-terminal kinase 3) was unaffected by the K285A/R286A or L295A mutations. These results suggest that amino acids important for self-association of β-arrestin 2 also play an important role in the interaction with both the β 2 -adrenoceptor and the ERK1/2 MAPKs. Regulation of β-arrestin 2 self-association may therefore control β-arrestin 2-mediated β 2 -adrenoceptor-ERK1/2 MAPK signalling.

Opioid agonists have a broad range of effects on cells of the immune system, including modulation of the inflammatory response, and opioid and chemokine receptors are co-expressed by many white cells. Hetero-oligomerization of the human DOP opioid and chemokine CXCR2 receptors could be detected following their co-expression by each of co-immunoprecipitation, three different resonance energy transfer techniques and the construction of pairs of individually inactive but potentially complementary receptor G-protein α subunit fusion proteins. Although DOP receptor agonists and a CXCR2 antagonist had no inherent affinity for the alternative receptor when either receptor was expressed individually, use of cells that expressed a DOP opioid receptor construct constitutively, and in which expression of a CXCR2 receptor construct could be regulated, demonstrated that the CXCR2 antagonist enhanced the function of DOP receptor agonists only in the presence of CXCR2. This effect was observed for both enkephalin- and alkaloid-based opioid agonists, and the effective concentrations of the CXCR2 antagonist reflected CXCR2 receptor occupancy. Entirely equivalent results were obtained in cells in which the native DOP opioid receptor was expressed constitutively and in which expression of the isolated CXCR2 receptor could be induced. These results indicate that a CXCR2 receptor antagonist can enhance the function of agonists at a receptor for which it has no inherent direct affinity by acting as an allosteric regulator of a receptor that is a heterodimer partner for the CXCR2 receptor. These results have novel and important implications for the development and use of small-molecule therapeutics.

β 2 -ARs (β 2 -adrenoceptors) become desensitized rapidly upon recruitment of cytosolic β-arrestin. PDE4D5 (family 4 cAMP-specific phosphodiesterase, subfamily D, isoform 5) can be recruited in complex with β-arrestin, whereupon it regulates PKA (cAMP-dependent protein kinase) phosphorylation of the β 2 -AR. In the present study, we have used novel technology, employing a library of overlapping peptides (25-mers) immobilized on cellulose membranes that scan the entire sequence of β-arrestin 2, to define the interaction sites on β-arrestin 2 for binding of PDE4D5 and the cognate long isoform, PDE4D3. We have identified a binding site in the β-arrestin 2 N-domain for the common PDE4D catalytic unit and two regions in the β-arrestin 2 C-domain that confer specificity for PDE4D5 binding. Alanine-scanning peptide array analysis of the N-domain binding region identified severely reduced interaction with PDE4D5 upon R26A substitution, and reduced interaction upon either K18A or T20A substitution. Similar analysis of the β-arrestin 2 C-domain identified Arg 286 and Asp 291 , together with the Leu 215 –His 220 region, as being important for binding PDE4D5, but not PDE4D3. Transfection with wild-type β-arrestin 2 profoundly decreased isoprenaline-stimulated PKA phosphorylation of the β 2 -AR in MEFs (mouse embryo fibroblasts) lacking both β-arrestin 1 and β-arrestin 2. This effect was negated using either the R26A or the R286A mutant form of β-arrestin 2 or a mutant with substitution of an alanine cassette for Leu 215 –His 220 , which showed little or no PDE4D5 binding, but was still recruited to the β 2 -AR upon isoprenaline challenge. These data show that the interaction of PDE4D5 with both the N- and C-domains of β-arrestin 2 are essential for β 2 -AR regulation.

The orexin-1 receptor interacts with β-arrestin-2 in an agonist-dependent manner. In HEK-293T cells, these two proteins became co-internalized into acidic endosomes. Truncations from the C-terminal tail did not prevent agonist-induced internalization of the orexin-1 receptor or alter the pathway of internalization, although such mutants failed to interact with β-arrestin-2 in a sustained manner or produce its co-internalization. Mutation of a cluster of three threonine and one serine residue at the extreme C-terminus of the receptor greatly reduced interaction and abolished co-internalization of β-arrestin-2–GFP (green fluorescent protein). Despite the weak interactions of this C-terminally mutated form of the receptor with β-arrestin-2, studies in wild-type and β-arrestin-deficient mouse embryo fibroblasts confirmed that agonist-induced internalization of this mutant required expression of a β-arrestin. Although without effect on agonist-mediated elevation of intracellular Ca 2+ levels, the C-terminally mutated form of the orexin-1 receptor was unable to sustain phosphorylation of the MAPKs (mitogen-activated protein kinases) ERK1 and ERK2 (extracellular-signal-regulated kinases 1 and 2) to the same extent as the wild-type receptor. These studies indicate that a single cluster of hydroxy amino acids within the C-terminal seven amino acids of the orexin-1 receptor determine the sustainability of interaction with β-arrestin-2, and indicate an important role of β-arrestin scaffolding in defining the kinetics of orexin-1 receptor-mediated ERK MAPK activation.

Post-translational thio-acylation of a fusion protein between the α 2A -adrenoceptor and the α subunit of the G protein G o1 is both dynamic and regulated by agonist binding. Incorporation of [ 3 H]palmitate into the fusion protein was reduced substantially in the presence of the agonist adrenaline. This was dependent on the concentration of adrenaline and correlated with occupancy of the ligand binding site. Both the receptor and G-protein elements of the fusion construct incorporated [ 3 H]palmitate but this occurred more rapidly for the G-protein element and regulation of acylation by the agonist occurred only for the G protein. The kinetics of de-palmitoylation of the α 2A -adrenoceptor–Gα o1 fusion were accelerated markedly by agonist. Again, this reflected modulation of the G protein but not of the receptor. Agonist-induced regulation of the kinetics of thio-acylation of the G protein was abolished, however, in a mutant unable to bind guanosine 5′-[γ-[ 35 S]thio]triphosphate ([ 35 S]GTP[S]) in response to adrenaline. Despite the dynamic nature of the post-translational acylation and its regulation by agonist, the ability of adrenaline to activate the G protein, monitored by stimulation of the binding of [ 35 S]GTP[S] to such fusion constructs, was unaffected by the palmitoylation potential of either the receptor or G-protein element.

Homo- and hetero-oligomerization of G-protein-coupled receptors (GPCRs) were examined in HEK-293 cells using two variants of bioluminescence resonance energy transfer (BRET). BRET 2 (a variant of BRET) offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared with traditional BRET. Previously recorded homo-oligomerization of the human δ-opioid receptor was confirmed using BRET 2 . Homo-oligomerization of the κ-opioid receptor was observed using both BRET techniques. Both homo- and hetero-oligomers, containing both δ- and κ-opioid receptors, were unaffected by the presence of receptor ligands. BRET detection of opioid receptor homo- and hetero-oligomers required expression of 50000–100000 copies of the receptor energy acceptor construct per cell. The effectiveness of δ—κ-opioid receptor hetero-oligomer formation was as great as for homomeric interactions. The capacity of the two opioid receptors to form oligomeric complexes with the β 2 -adrenoceptor was also assessed. Although such interactions were detected, at least 250000 copies per cell of the energy acceptor were required. Requirement for high levels of receptor expression was equally pronounced in attempts to measure hetero-oligomer formation between the κ-opioid receptor and the thyrotropin-releasing hormone receptor-1. These studies indicate that constitutively formed homo- and hetero-oligomers of opioid receptor subtypes can be detected in living cells containing less than 100000 copies of the receptors. However, although hetero-oligomeric interactions between certain less closely related GPCRs can be detected, they appear to be of lower affinity than homo- or hetero-oligomers containing closely related sequences. Interactions recorded between certain GPCR family members in heterologous expression systems are likely to be artefacts of extreme levels of overexpression.

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.

Direct measures of G-protein activation based on guanine nucleotide exchange and hydrolysis are frequently impossible to monitor for receptors which interact predominantly with G s α. An isolated FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)-epitope-tagged human IP prostanoid receptor and fusion proteins generated between this form of the receptor and the α subunits of its cognate G-protein G s , G i1 , a G-protein which it fails to activate in co-expression studies, and a chimaeric G i1 -G s 6 (a form of G i1 in which the C-terminal six amino acids were replaced with the equivalent sequence of G s ) were stably expressed in HEK293 cells. These were detected by [ 3 H]ligand-binding studies and by immunoblotting with both an anti-FLAG antibody and with appropriate antisera to the G-proteins. Each construct displayed similar affinity to bind the agonist iloprost. Iloprost stimulated adenylate cyclase activity in clones expressing both IP prostanoid receptor and the IP prostanoid receptor-G s α fusion protein, and both constructs were shown to interact with and activate endogenously expressed G s α. Addition of iloprost to membranes of cells expressing the isolated receptor resulted in a small stimulation of high-affinity GTPase activity. Iloprost produced no stimulation of GTPase activity which could be attributed to the IP prostanoid receptor-G i1 α fusion. However, the fusion proteins containing either G s α or G i1 -G s 6α produced substantially greater stimulation of GTPase activity than the isolated IP prostanoid receptor. Treatment of cells expressing the IP prostanoid receptor-G i1 -G s 6α fusion protein with a combination of cholera and pertussis toxins allowed direct measurement of agonist activation of the receptor-linked G-protein. Normalization of such results for levels of expression of the IP prostanoid receptor constructs demonstrated a 5-fold higher stimulation of GTPase activity when using the G s α-containing fusion protein and a 9-fold improvement when using the fusion protein containing G i1 -G s 6α to detect G-protein activation compared with expression of the isolated receptor.

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 G q α/G 11 α, identified by indirect immunofluorescence, was monitored concurrently. Addition of thyrotropin-releasing hormone resulted in rapid separation of TRHR-1-GFP and G q α/G 11 α signals as the receptor was internalized. This situation persisted for more than an hour. At longer time periods a fraction of the cellular G q α/G 11 α was also internalized, although much of the G q α/G 11 α immunoreactivity remained associated with the plasma membrane. Parallel experiments, in which the cellular distribution of TRHR-1-GFP and G q α/G 11 α 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 G q α/G 11 α 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.

The α 1 -adrenergic agonist phenylephrine stimulated phospholipase D (PLD) activity in Rat 1 fibroblasts transfected to express either the wild-type hamster α 1B -adrenoceptor or a constitutively active mutant (CAM) form of this receptor. The EC 50 for agonist stimulation of PLD activity was substantially lower at the CAM receptor than at the wild-type receptor as previously noted for phenylephrine stimulation of phosphoinositidase C activity. Sustained treatment of cells expressing the CAM α 1B -adrenoceptor with phentolamine resulted in a marked up-regulation in levels of this receptor with half-maximal effects produced within 24 h and with an EC 50 of approx. 40 nM. Such an up-regulation could be produced with a range of other ligands generally viewed as α 1 -adrenoceptor antagonists but equivalent treatment of cells expressing the wild-type α 1B -adrenoceptor was unable to mimic these effects. After sustained treatment of the CAM α 1B -adrenoceptor expressing cells with phentolamine, basal PLD activity was increased and phenylephrine was now able to stimulate PLD activity to greater levels than in vehicle-treated CAM α 1B -adrenoceptor-expressing cells. The EC 50 for phenylephrine stimulation of PLD activity was not altered, however, by phentolamine pretreatment and the associated up-regulation of the receptor. After phentolamine-induced up-regulation of basal PLD activity, a range of α 1 -antagonists were shown to possess the characteristics of inverse agonists of the CAM α 1B -adrenoceptor as they were able to substantially decrease the elevated basal PLD activity.

The regulation of mitogenic signalling pathways by G-protein-coupled receptors has been studied in Rat-1 fibroblasts stably transfected with the murine δ opioid receptor. We showed recently that stimulation of this receptor led to the activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinase [Burt, Carr, Mullaney, Anderson and Milligan (1996) Biochem. J. 320 , 227–235]. The present study has examined the role of the ribosomal S6 kinase p70 s6k in mitogenic signalling by the δ opioid receptor. Treatment of Rat-1 fibroblasts expressing this receptor with the synthetic enkephalin [ d -Ala, d -Leu]-enkephalin (DADLE) led to a dose-dependent increase in p70 s6k enzyme activity. Activation of p70 s6k was dependent on the level of δ opioid receptor expressed and was sustained above basal levels for several hours. Immunoblotting revealed that p70 s6k was subject to increased phosphorylation, the extent of which coincided temporally with enzyme activation. Activation of p70 s6k by DADLE, but not by platelet-derived growth factor, was blocked by pretreatment of cells with pertussis toxin. Activation of p70 s6k was also partly blocked by wortmannin, indicating that phosphoinositide 3-OH kinase is required for full activation of p70 s6k by opioid receptor agonists. Activation of the δ opioid receptor in transfected cells led to increased DNA synthesis. This increase was prevented by rapamycin, which also completely blocked activation of p70 s6k by DADLE. In addition, prevention of the activation of p42 and p44 MAP kinases also blocked the induction of DNA synthesis by DADLE. These results suggest that the activation of both MAP kinases and p70 s6k might be crucial to the induction of mitogenic responses by G i -linked receptors such as the δ opioid receptor.

A fusion protein was generated between the porcine α 2A -adrenoceptor and a pertussis-toxin-insensitive (Cys 351 → Gly) variant of the α subunit of G i1 α by direct in-frame fusion of the N-terminus of the G-protein to the C-terminus of the receptor. The fusion protein could be transiently expressed to high levels in COS-7 cells. Addition of the α 2 -adrenoceptor agonist 5-bromo- N -(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) to membranes of pertussis-toxin-treated transfected cells resulted in a concentration-dependent stimulation of high-affinity GTPase activity. V max estimations for the GTPase activity demonstrated an induced catalytic-centre activity of 2.0±0.2 min -1 for G i1 α when the α 2A -adrenoceptor was maximally stimulated by UK14304 with a K m for GTP of 0.37±0.07 μ M. Co-expression of excess β 1 γ 2 along with the α 2A -adrenoceptor-G i1 α fusion protein resulted in greater maximal UK14304-induced stimulation of high-affinity GTPase activity (2.1±0.2-fold) without alteration in agonist EC 50 . These studies demonstrate the functionality of the fusion construct, its capacity to interact with βγ complex and its utility in measuring agonist regulation of the catalytic-centre activity of GTP by a receptor-associated G-protein.

The α 2A -adrenoceptor is the prototypic example of the family of G-protein-coupled receptors which function by activation of ‘G i -like’ pertussis toxin-sensitive G-proteins. A number of members of this subfamily of G-proteins are often co-expressed in a single cell type. To examine the interaction of this receptor with individual G i -family G-proteins the porcine α 2A -adrenoceptor was transiently transfected into COS-7 cells either alone or with each of wild-type G i 1α, G i 2α and G i 3α or mutations of each of these G-proteins in which the cysteine residue which is the target for pertussis toxin-catalysed ADP-ribosylation was exchanged for a glycine residue. The α 2 -adrenoceptor agonist UK14304 stimulated both high-affinity GTPase activity and the binding of guanosine 5ƀ-[γ- 35 thio]-triphosphate (GTP[ 35 S]), when expressed without any additional G-protein. These effects were greatly reduced by pretreatment of the cells with pertussis toxin. Co-expression of each of the wild-type G i -like G-protein α-subunits resulted in enhanced agonist activation of the cellular G-protein population which was fully prevented by pretreatment with pertussis toxin. Co-expression of the receptor along with the cysteine-to-glycine mutations of G i 1α, G i 2α and G i 3α resulted in agonist stimulation of these G-proteins, which was as great as that of the wild type proteins, but now the agonist stimulation produced over that due to the activation of endogenously expressed G i -like G-proteins was resistant to pertussis toxin treatment. The Cys → Gly mutations of G i 1α, G i 2α and G i3 α were each also able to limit agonist-mediated stimulation of adenylate cyclase activity. The degree of agonist-mediated activation of the pertussis toxin-resistant mutant of G i 1a was correlated highly both with the level of expression of this G-protein and with the level of expression of the α 2A -adrenoceptor. Half-maximal stimulation of high-affinity GTPase activity of the Cys → Gly mutants of G i 1α, G i 2α and G i 3α required 10Ő15-fold higher concentrations of agonist than did stimulation of their wild-type counterparts, consistent with a model in which the affinity of functional interactions of the α 2A -adrenoceptor with the wild-type G-protein is greater than with the pertussis toxin-resistant mutant G-protein.

Heterotrimeric G-proteins have been found in eukaryotic cells, from yeast to humans, but have received little attention, to date, with respect to parasitic organisms. We now present the first report of the characterization of heterotrimeric G-proteins expressed in a filarial nematode, Acanthocheilonema viteae . Using a combination of (i) affinity labelling with [α- 32 P]GTP; (ii) ADP-ribosylation with cholera toxin and pertussis toxin; (iii) Western blotting with a panel of anti-G-protein antibodies; and (iv) reverse transcriptase-PCR with degenerate G-protein oligonucleotide primers followed by hybridization analysis using oligonucleotides specific for individual G-protein subunits, we demonstrate that adult A. viteae expresses homologues of the β1-and/or β2-like subunits and α-subunits of the G s , G i , G q and G 12 subfamilies found in mammals. The role which these G-proteins may play in the biology of the organism is discussed.

Rat 1 fibroblasts transfected to express either the wild-type hamster α 1B -adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid residues 288–294 to encode the equivalent region of the human β 2 -adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAMα 1B -adrenergic receptor was greater than for the wild-type receptor. The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAMα 1B -adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phosphate production was approx. 200-fold greater at the CAMα 1B -adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ET A receptor, displayed similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the α subunits of the phosphoinositidase C-linked, pertussis toxin-insensitive, G-proteins G q and G 11 in cells expressing either the wild-type or the CAMα 1B -adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAMα 1B -adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAMα 1B -adrenergic receptor than at the wild-type receptor. There were no detectable differences in the basal rate of G q α/G 11 α degradation between cells expressing the wild-type or the CAMα 1B -adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degradation of these G-proteins, with a greater effect at the CAMα 1B -adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAMα 1B -adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to G q and G 11 .

Rat-1 fibroblasts were transfected with a cDNA encoding the mouse Δ opioid receptor. Two separate clones, D2 (which expressed some 6 pmol of the receptor/mg of membrane protein) and DOE (which expressed some 0.2 pmol/mg of membrane protein), were examined in detail. With membranes from both clones, the opioid agonist [ D -Ala 2 ]leucine enkephalin (DADLE) caused stimulation of high-affinity GTPase activity and of the binding of guanosine 5´-[γ-[ 35 S]thio]triphosphate, and inhibition of forskolin-amplified adenylate cyclase activity. DADLE also induced phosphorylation and activation of both the p42 MAPK (42 kDa isoform) and p44 MAPK (44 kDa isoform) members of the mitogen-activated protein kinase (MAP kinase) family. All of these effects of DADLE were prevented in both clones by pretreatment of the cells with pertussis toxin. The maximal response that could be produced by DADLE in direct assays of G-protein activation were substantially greater in clone D2 than in clone DOE, but in both clones essentially full phosphorylation of both p42 MAPK and p44 MAPK could be achieved. EC 50 values for DADLE stimulation of GTPase activity and for activation of p44 MAPK were substantially lower in clone D2 than in clone DOE. Moreover, in both clones the EC 50 value for DADLE stimulation of p44 MAPK was substantially lower than that for stimulation of GTPase activity, and the Hill coefficients for agonist activation of p44 MAPK ( h > 1) displayed marked co-operativity whereas those for G-protein activation did not ( h 0.8–1.0). DADLE activation of p44 MAPK showed more sustained kinetics in clone D2 than in clone DOE. By contrast, lysophosphatidic acid, acting at an endogenously expressed G-protein-coupled receptor, also activated p44 MAPK in both clones in a pertussis toxin-sensitive manner, but both the kinetics and the concentration–response curve for activation of p44 MAPK by this ligand were similar. As with other systems, maintained cellular levels of a cAMP analogue prevented the effects of both G-protein-coupled receptors on activation of p44 MAPK . These results demonstrate for the first time that an opioid receptor, at least when expressed in Rat-1 fibroblasts, is able to initiate activation of the MAP kinase cascade in a G i -dependent manner, and show that only a very small proportion of the cellular G i population is required to be activated to result in full phosphorylation of the p42 MAPK and p44 MAPK MAP kinases.

Stable cell lines, derived from NG108-15 cells and transfected to express both the β 2 -adrenoceptor and adenylate cyclase type II, were produced and examined. The absence of adenylate cyclase type II in the parental cells and its presence in these clones was demonstrated by reverse transcriptase-PCR. Total cellular levels of adenylate cyclase were increased in a number of clones between 3- and 8-fold, as assessed by guanine nucleotide-stimulated specific high-affinity binding of [ 3 H]forskolin to cellular membranes. Basal adenylate cyclase activity was markedly elevated compared with a clone expressing similar levels of the β 2 -adrenoceptor in the absence of adenylate cyclase type II. Each of NaF, forskolin and guanosine 5´-[β,γ-imido]triphosphate (a poorly hydrolysed analogue of GTP) produced substantially higher levels of adenylate cyclase activity in membranes of the clones positive for expression of adenylate cyclase type II than was achieved with the parental cells. Both isoprenaline, acting at the introduced β 2 -adrenoceptor, and iloprost, acting at the endogenously expressed IP prostanoid receptor, stimulated adenylate cyclase activity to much higher levels in the clones expressing adenylate cyclase type II compared with the clone lacking this adenylate cyclase; however, the concentration–effect curves for adenylate cyclase stimulation by these two agonists were not different between parental cells and clones overexpressing adenylate cyclase type II. A maximally effective concentration of the β-adrenoceptor partial agonist ephedrine displayed similar intrinsic activity and potency to stimulate adenylate cyclase in membranes of clones both with and without adenylate cyclase type II. Both secretin and 5´- N -ethylcarboxamidoadenosine (acting at an endogenous A 2 adenosine receptor) were also able to produce substantially greater maximal activations of adenylate cyclase in the clones expressing excess adenylate cyclase type II, without alterations in agonist intrinsic activity or potency. These results demonstrate that the maximal output of the stimulatory arm of the adenylate cyclase cascade can be increased by increasing total levels of adenylate cyclase in the genetic background of NG108-15 cells.

The significance of G i proteins for the physiological desensitization phenomena observed in brown-fat cells from cold-acclimated hamsters was investigated. For this purpose, pertussis toxin (the inhibitor of G i function) was injected into control and cold-acclimated hamsters. After 3 days the thermogenic response to noradrenaline injection was monitored in the intact animals. It was found that the pertussis-toxin pretreatment did not affect the thermogenic response to noradrenaline. Nonetheless, the pertussis toxin pretreatment had a dramatic effect on the noradrenaline-sensitivity of isolated brown-fat cells (measured the following day as the respiratory response): a 250-fold-increased sensitivity to noradrenaline was observed in cells from control animals that had been pertussis-toxin pretreated. However, only a 20-fold increase was observed in cells from cold-acclimated hamsters, implying a lower complement of the G i system in these cells. Therefore the content of G i proteins was determined by quantitative immunoblotting of purified plasma-membrane proteins. Cold acclimation resulted in a nearly 50% reduction in the content of G i 1α and G i 2α, as well as of the β-subunit, both when expressed on a protein basis and when related to the content of forskolin-stimulated adenylyl cyclase; when expressed per unit of [ 3 H]ouabain-binding (Na + /K + -ATPase), the reduction was even higher. In view of the magnitude of the pertussis-toxin effect, it was concluded that G i proteins must play a substantial role in the regulation of the response of brown-fat cells to noradrenaline. As the capacity of the G i pathway is reduced rather than augmented during cold acclimation, G i activity cannot be responsible for the desensitization to noradrenaline observed in cells from cold-acclimated animals. However, the reduced G i content may explain the earlier observed desensitization to adenosine that occurs after acclimation to cold.