Sandra Siehler

Regulation of RhoGEF proteins by G12/13-coupled receptors

G protein‐coupled receptors (GPCRs) represent a large family of seven transmembrane receptors, which communicate extracellular signals into the cellular lumen. The human genome contains 720–800 GPCRs, and their diverse signal characteristics are determined by their specific tissue and subcellular expression profiles, as well as their coupling profile to the various G protein families (Gs, Gi, Gq, G12). The G protein coupling pattern links GPCR activation to the specific downstream effector pathways. G12/13 signalling of GPCRs has been studied only recently in more detail, and involves activation of RhoGTPase nucleotide exchange factors (RhoGEFs). Four mammalian RhoGEFs regulated by G12/13 proteins are known: p115‐RhoGEF, PSD‐95/Disc‐large/ZO‐1 homology‐RhoGEF, leukemia‐associated RhoGEF and lymphoid blast crisis‐RhoGEF. These link GPCRs to activation of the small monomeric GTPase RhoA, and other downstream effectors. Misregulated G12/13 signalling is involved in multiple pathophysiological conditions such as cancer, cardiovascular diseases, arterial and pulmonary hypertension, and bronchial asthma. Specific targeting of G12/13 signalling‐related diseases of GPCRs hence provides novel therapeutic approaches. Assays to quantitatively measure GPCR‐mediated activation of G12/13 are only emerging, and are required to understand the G12/13‐linked pharmacology. The review gives an overview of G12/13 signalling of GPCRs with a focus on RhoGEF proteins as the immediate mediators of G12/13 activation.

[125I]Tyr10-cortistatin14 labels all five somatostatin receptors

The recently cloned rat preprocortistatin, which shows homology to the preprosomatostatin peptide, is thought to be enzymatically cleaved to cortistatin14 (CST14) similarly to somatostatin14 (SRIF14). High structural similarity of cortistatin14 compared to SRIF14 suggested binding properties to somatostatin receptors similar to SRIF14. In the present study, we expressed stably the five human somatostatin receptor subtypes (hsst1–hsst5) in CCL39 cells (Chinese hamster lung fibroblast cells). The receptors were labelled with an iodinated analogue of CST14 ([125I]Tyr10-cortistatin14, [125I]Tyr10-CST) to establish the pharmacological profile of hsst1–hsst5 sites labelled with [125I]Tyr10-CST. In parallel, [Leu8,D-Trp22,125I-Tyr25]-SRIF28 ([125I]LTT-SRIF28) was used as a control at the five recombinant SRIF receptors stably expressed in CCL39 cells. High affinity [125I]Tyr10-CST binding could be demonstrated to all five recombinant somatostatin receptor subtypes. The pKd (–log mol/l) and Bmax values (fmol/mg) for hsst1–5 receptors were: 10.02±0.04, 220±30; 9.45±0.09, 340±70; 10.06±0.11, 340±50; 9.67±0.14, 340±110 and 10.33±0.03, 5630±330, respectively. The pharmacological profiles determined with [125I]Tyr10-CST and [125I]LTT-SRIF28 were very similar at every receptor studied. These data suggest that cortistatin and somatostatin have similar high affinity for SRIF receptors. None of the receptors showed marked selectivity for either CST14/CST17 or the somatostatins. In conclusion, the data show that cortistatin and somatostatin have very similar high affinity to all five recombinant somatostatin receptors. It remains to be seen whether there are specific receptors which bind only somatostatins or cortistatins.

Distribution and characterisation of somatostatin receptor mRNA and binding sites in the brain and periphery

The distribution and nature of (somatostatin) SRIF receptors and receptor mRNAs was studied in the brain and periphery of various laboratory animals using in situ hybridisation, autoradiography and radioligand binding. The messenger RNA (mRNA) expression of SRIF receptors msst1, msst2, msst3, msst4 and msst5 was studied in the adult mouse brain by in situ hybridisation histochemistry using specific oligonucleotide probes and compared to that of adult rats. As observed in rat brain, sst3 receptor mRNA is prominently expressed across the mouse brain, although equivalent binding has not yet been identified in situ. Sst1 and sst2 receptor mRNA expression, was prominent and again comparable to that observed in rat brain, whereas sst4 and especially sst5 receptor mRNA show comparatively low levels, although the former appears to be widely distributed while the latter could only be identified in a few nuclei. Altogether, the data are compatible with current knowledge, i.e. sst1 and sst2 receptor mRNA is prominent (both receptors have been functionally identified in the brain and for sst2 in the periphery), sst3 mRNA is highly expressed but in the absence of any functional correlate remains elusive. The expression of sst4 mRNA is comparatively low (especially when compared to what is seen in the lung, where high densities of sst4 receptors are present) and it remains to be seen whether sst5 receptor mRNA, which is confined to a few nuclei, will play a role in the brain, keeping in mind that high levels are found in the pituitary. Radioligand binding studies were performed in CCL39 cells expressing the five human recombinant receptors and compared to binding in membranes of rat cerebral cortex with [125I]Tyr11-SRIF14 which in the presence of 120 mM labels primarily sst1 receptor as suggested by the better correlation hsst1 and similar rank order of potency. The profile of [125I]Tyr3-octreotide labelled sites in rat cortex correlates better with recombinant sst2 than sst3 or sst5 binding profiles. Finally, [125I]LTT-SRIF28-labelled sites in rat lung express a sst4 receptor profile in agreement with previous findings. SRIF receptor autoradiography was performed in the brain and peripheral tissue of rat and/or guinea-pig using a number of ligands known to label recombinant SRIF receptors: [125I]LTT-SRIF28, [125I]CGP 23996, [125I]Tyr10-CST, or [125I]Tyr3-octreotide. Although, [125I]Tyr10-CST has been shown to label all five recombinant SRIF receptors, it is apparent that this radioligand is not useful for autoradiographic studies. By contrast, the other three ligands show good signal to noise ratios in rat or guinea-pig brain, rat lung, rat pancreas, or guinea-pig ileum. In most tissues, [125I]Tyr3-octreotide represents a prominent part of the binding (when compared to [125I]LTT-SRIF28 and [125I]CGP 23996), suggesting that sst2 receptors are strongly expressed in most tissues; it is only in rat lung that [125I]LTT-SRIF28 and [125I]CGP 23996 show marked binding, whereas [125I]Tyr3-octreotide does apparently label no sites, in agreement with the sole presence of sst4 receptors in this tissue.

Pharmacological profile of somatostatin and cortistatin receptors

Somatostatin (SRIF) and cortistatin (CST) are two endogenous peptides with high sequence similarities that act as hormones/neurotransmitters both in the CNS and the periphery; their genes although distinct result from gene duplication. Their receptors appear to be common, since the five known SRIF receptors (sst1-sst5) have similar subnanomolar affinity for SRIF and CST, whether the short (SRIF-14, CST-14, CST-17) or the long versions (SRIF-28, CST-29) of the peptides. Whether CST targets specific receptors not shared by SRIF, is still debated: MrgX2 has been described as a selective CST receptor, with submicromolar affinity for CST but devoid of affinity for SRIF; however the distribution of CST and MrgX2 is largely different, and there is no MrgX2 in rodents. A similar situation arises with the GHS receptor GHS-R1a, which displays some preferential affinity for CST over SRIF, but for which there is no evidence that it is activated by CST in vivo. In both cases, one may argue that submicromolar affinity is not the norm of a GPCR for its endogenous neuropeptide. On the other hand, all receptors known to bind SRIF have similar high affinity for CST and both peptides act as potent agonists at the sst1-sst5 receptors, whichever transduction pathway is considered. In addition, [(125)I][Tyr(10)]CST(14) labels sst1-sst5 receptors with subnanomolar affinity, and [(125)I][Tyr(10)]CST(14) binding in the brain is overlapping with that of [(125)I][Tyr(0)]SRIF(14). The functional differences reported that distinguish CST from SRIF, have not been explained convincingly and may relate to ligand-driven transductional selectivity, and other complicating factors such as receptor dimerisation, (homo or heterodimerisation), and/or the influence of accessory proteins (GIPs, RAMPS), which remain to be studied in more detail.

Cell-based assays in GPCR drug discovery.

G protein‐coupled receptors (GPCRs) transmit extracellular signals into the intracellular space, and play key roles in the physiological regulation of virtually every cell and tissue. Characteristic for the GPCR superfamily of cell surface receptors are their seven transmembrane‐spanning α‐helices, an extracellular N terminus and intracellular C‐terminal tail. Besides transmission of extracellular signals, their activity is modulated by cellular signals in an auto‐ or transregulatory fashion. The molecular complexity of GPCRs and their regulated signaling networks triggered the interest in academic research groups to explore them further, and their drugability and role in pathophysiology triggers pharmaceutical research towards small molecular weight ligands and therapeutic antibodies. About 30% of marketed drugs target GPCRs, which underlines the importance of this target class. This review describes current and emerging cellular assays for the ligand discovery of GPCRs.

Structural and Compositional Determinants of Cortistatin Activity

Cortistatin‐14 (CST‐14) is a putative novel neuropeptide that shares 11 of its 14 residues with somatostatin‐14 (SRIF‐14), yet its effects on sleep physiology, locomotor behavior and hippocampal function are different from those of somatostatin. We studied the structural basis for cortistatins distinct biological activities. As with SRIF‐14, CST‐14 does not show any preferred conformation in solution, as determined by circular dichroism and nuclear magnetic resonance. Synthetic cortistatin analogs were designed and synthesized based on the cyclic structure of octreotide. Biological assays were carried out to determine their binding affinities to five somatostatin receptors (sst1‐5) and their ability to produce changes in locomotor activity and to modulate hippocampal physiology and sleep. The results show that the compound with N‐terminal proline and C‐terminal lysine amide exhibits cortistatin‐like biological activities, including reduction of population spike amplitudes in the hippocampal CA1 region, decrease in locomotor activity and enhancement of slow‐wave sleep 2. These findings suggest that both proline and lysine are necessary for cortistatin binding to its specific receptor. J. Neurosci. Res. 56:611–619, 1999. 

Cloning, expression and pharmacological characterisation of the mouse somatostatin sst5 receptor

Abstract The mouse somatostatin (somatotropin release inhibiting factor, SRIF) sst 5 receptor coding sequence was cloned from a mouse BALB/c genomic library. It shows 97% and 81% homology with the corresponding rat and human receptors, respectively. The msst 5 receptor messenger RNA (mRNA) is present at low levels in the adult mouse brain, with significant expression in a few nuclei only, e.g. in the septum (lateral septal nuclei) or the amygdala (medial amygdaloid nucleus); very few signals were observed in the mesencephalon, metencephalon, and myelencephalon (except the dorsal motor nucleus of the vagus nerve). The msst 5 receptor was stably expressed in the hamster fibroblast cell line CCL39-SRE-Luci, which harbours the luciferase reporter gene driven by the serum responsive element. [ 125 I]LTT-SRIF-28 ([Leu 8 , D-Trp 22 , 125 I-Tyr 25 ]-SRIF-28), [ 125 I]Tyr 10 -CST, [ 125 I]CGP 23996, and [ 125 I]Tyr 3 -octreotide labelled msst 5 receptors with high affinity (pK d values: 11.0, 10.15, 9.75 and 9.43) and in a saturable manner, but defined different Bmax values: 697, 495, 540 and 144 fmoles/mg, respectively. [ 125 I]LTT-SRIF-28-labelled sites displayed the following rank order: SRIF-28> rCST-14> somatuline > CGP-23996= SRIF-14= octreotide, whereas [ 125 I]Tyr 3 -octreotide-labelled sites displayed a different profile: octreotide > SRIF-28> rCST-14= somatuline > SRIF-14> CGP-23996. The pharmacological profiles determined with [ 125 I]LTT-SRIF-28, [ 125 I]CGP 23996 and [ 125 I]Tyr 10 -CST correlated highly significantly (r 2 =0.88–0.99), whereas [ 125 I]Tyr 3 -octreotide binding was rather divergent (r 2 =0.77). Also, human and mouse sst 5 receptor profiles are very different, e.g. r 2 =0.385 for [ 125 I]Tyr 10 -CST and r 2 =0.323 for [ 125 I]LTT-SRIF-28-labelled sites. Somatostatin induces expression of luciferase reporter gene in CCL39-SRE-Luci cells. The profile was consistent with a msst 5 receptor-mediated effect although apparent potency in the luciferase assay was much reduced compared to radioligand binding data: Octreotide = SRIF-28> rCST-14= SRIF-14= CGP-23996. Octreotide, SRIF-28, BIM23052 and D Tyr Cyanamid 154806 behaved as full or nearly full agonists in comparison to SRIF-14, whereas the other compounds had relative efficacies of 40 to 70%. The present study shows that agonists radioligands define apparently different receptor populations in terms of number of sites and pharmacological profile in cells expressing a single recombinant receptor. These variations suggest that the conformation of the ligand receptor complex may vary depending on the agonist. Further, the msst 5 receptor, although primarily coupled to Gi/Go proteins, is able to stimulate luciferase gene expression driven by the serum responsive element. Finally, it is suggested that putative sst 2 selective agonists e.g. octreotide, RC160 or BIM23027 show similar or higher potency at msst 5 receptors than SRIF-14.

Characterisation of the fish sst3 receptor, a member of the SRIF1 receptor family : atypical pharmacological features

The first cloned non-mammalian somatostatin (somatostatin release-inhibiting factor = SRIF) receptor previously obtained from the teleost fish Apteronotus albifrons and generically named somatostatin receptor 3 (fsst3), was stably expressed and characterised in Chinese hamster lung fibroblast (CCL39) cells. Radioligand binding studies were performed with four radioligands selective for SRIF receptors in CCL39 cells expressing the fsst3 receptors; [125I]LTT-SRIF28 ([Leu8, D-Trp22, 125I-Tyr25]-SRIF28), [125I]Tyr10-cortistatin, [125I]CGP 23996, and [125I]Tyr3-octreotide labelled the fsst3 receptor with high affinity (pKd values: 10.47, 10.87, 9.59 and 9.57) and in a saturable manner, but defined different Bmax values; 4500, 4000, 3400 and 1500 fmol/mg, respectively. The affinities of SRIF peptides and analogues determined for fsst3 receptors displayed the following rank order of potency: seglitide = SRIF25 > SRIF14 = SRIF28 > cortistatin 14 > BIM 23014 > RC160 = L361,301 = octreotide > or = BIM 23052 > or = L362,855 > CGP23996 > BIM 23056 > BIM 23030 = cycloantagonist > SRIF22. The pharmacological profiles determined with [125I]LTT-SRIF28, [125I]CGP 23996 and [125I]Tyr10-cortistatin correlated highly significantly (r = 0.96-0.99), whereas [125I]Tyr3-octreotide binding was rather divergent (r = 0.78-0.81). Further, [125I]Tyr3-octreotide- and [125I]CGP 23996-labelled sites showed higher affinity for the various peptides than [125I]LTT-SRIF28 and [125I]Tyr10-cortistatin-labelled sites, although there were exceptions. [125I]LTT-SRIF28-binding to fsst3 receptors and human sst1-5 receptors was compared; the fsst3 binding profile correlated better with the hsst5- than with the hsst3 receptor profile. SRIF inhibited potently forskolin-stimulated adenylate cyclase activity in fsst3 transfected CCL39 cells; this effect was blocked by pertussis toxin, suggesting coupling of the fsst3 receptor to Gialpha and/or Goalpha. [125I]LTT-SRIF28 binding was detected in fish brain, liver, heart, spleen, and stomach, but not in gut. The pharmacological profile of [125I]LTT-SRIF28-labelled sites in brain, but not in liver, correlated significantly with the recombinant fsst3 receptor, in agreement with expression of the fsst3 receptor gene found by RT-PCR in the brain. However, biphasic binding curves obtained with two SRIF-analogues in brain, as well as the distinct pharmacological profile of the liver SRIF receptor, suggest the existence of several yet to be defined SRIF receptor subtypes in fish. The present data demonstrate that the recombinantly expressed fsst3 receptor has a pharmacological profile compatible with that of a SRIF1 receptor, although the rank order of affinity of fsst3 is closer to that of hsst5 than hsst3 receptors, as may be found when comparing very distantly related species. The fsst3 receptor expressed in CCL39 cells, is negatively coupled to adenylate cyclase activity via pertussis toxin-sensitive G-proteins, like mammalian sst3 receptors. Radioligand binding performed with fish tissue suggests the presence of a native sst3 receptor in brain as well as other yet to be defined SRIF receptor subtypes.

Reversible translocation of p115‐RhoGEF by G12/13‐coupled receptors

G protein‐coupled receptors (GPCRs) are important targets for medicinal agents. Four different G protein families, Gs, Gi, Gq, and G12, engage in their linkage to activation of receptor‐specific signal transduction pathways. G12 proteins were more recently studied, and upon activation by GPCRs they mediate activation of RhoGTPase guanine nucleotide exchange factors (RhoGEFs), which in turn activate the small GTPase RhoA. RhoA is involved in many cellular and physiological aspects, and a dysfunction of the G12/13‐Rho pathway can lead to hypertension, cardiovascular diseases, stroke, impaired wound healing and immune cell functions, cancer progression and metastasis, or asthma. In this study, regulator of G protein signaling (RGS) domain‐containing RhoGEFs were tagged with enhanced green fluorescent protein (EGFP) to detect their subcellular localization and translocation upon receptor activation. Constitutively active Gα12 and Gα13 mutants induced redistribution of these RhoGEFs from the cytosol to the plasma membrane. Furthermore, a pronounced and rapid translocation of p115‐RhoGEF from the cytosol to the plasma membrane was observed upon activation of several G12/13‐coupled GPCRs in a cell type‐independent fashion. Plasma membrane translocation of p115‐RhoGEF stimulated by a GPCR agonist could be completely and rapidly reversed by subsequent application of an antagonist for the respective GPCR, that is, p115‐RhoGEF relocated back to the cytosol. The translocation of RhoGEF by G12/13‐linked GPCRs can be quantified and therefore used for pharmacological studies of the pathway, and to discover active compounds in a G12/13‐related disease context. J. Cell. Biochem. 104: 1660–1670, 2008.

Receptor-specific regulation of ERK1/2 activation by members of the “free fatty acid receptor” family

Context: The “free fatty acid receptors” (FFARs) GPR40, GPR41, and GPR43 regulate various physiological homeostases, and are all linked to activation of extracellular signal-regulated kinases (ERK)1/2. Objective: Investigation of coupling of FFARs to two other mitogen-activated protein kinases (MAPKs) sometimes regulated by G protein-coupled receptors (GPCRs), c-Jun N-terminal kinase (JNK) and p38MAPK, and characterization of signaling proteins involved in the regulation of FFAR-mediated ERK1/2 activation. Methods: FFARs were recombinantly expressed, cells challenged with the respective agonist, and MAPK activation quantitatively determined using an AlphaScreen SureFire assay. Inhibitors for signaling proteins were utilized to characterize ERK1/2 pathways. Results: Propionate-stimulated GPR41 strongly coupled to ERK1/2 activation, while the coupling of linoleic acid-activated GPR40 and acetate-activated GPR43 was weaker. JNK and p38MAPK were weakly activated by FFARs. All three receptors activated ERK1/2 fully or partially via Gi/o and Rac. PI3K was relevant for GPR40- and GPR41-mediated ERK1/2 activation, and Src was essential for GPR40- and GPR43-induced activation. Raf-1 was not involved in the GPR43-triggered activation. Conclusion: The results demonstrate a novel role of Rac in GPCR-mediated ERK1/2 signaling, and that GPCRs belonging to the same family can regulate ERK1/2 activation by different receptor-specific mechanisms.