High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor.

High throughput random mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the α1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each α1 mutant was co-transfected with wildtype ß1, δ, and ε subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either α-bungarotoxin or tubocurarine. Eight α1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that random high throughput mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation.

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 G(i/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.

HDL-like discs for assaying membrane proteins in drug discovery.

To broaden the use of the recombinant high-density lipoprotein (rHDL) approach to the characterization of lead compounds, we investigated the pharmacology of the human beta-2-adrenoceptor in nanolipid bilayers (rHDL) with a broad set of beta-adrenoceptor antagonists. To that end, we developed a homogeneous copper-chelate scintillation proximity binding assay (SPA) in order to compare receptor-ligand binding affinities before and after reconstitution into rHDLs. Our results clearly show that the beta-2-adrenoceptor reconstituted in rHDLs display the same pharmacology as that in cell membranes and that rHDLs can be used not only to measure affinities for a range of ligands but also to study binding kinetics.

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 (G(s), G(i), G(q), G(12)). The G protein coupling pattern links GPCR activation to the specific downstream effector pathways. G(12/13) 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 G(12/13) 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 G(12/13) signalling is involved in multiple pathophysiological conditions such as cancer, cardiovascular diseases, arterial and pulmonary hypertension, and bronchial asthma. Specific targeting of G(12/13) signalling-related diseases of GPCRs hence provides novel therapeutic approaches. Assays to quantitatively measure GPCR-mediated activation of G(12/13) are only emerging, and are required to understand the G(12/13)-linked pharmacology. The review gives an overview of G(12/13) signalling of GPCRs with a focus on RhoGEF proteins as the immediate mediators of G(12/13) activation.

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 alpha-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.

Reversible translocation of p115-RhoGEF by G(12/13)-coupled receptors.

G protein-coupled receptors (GPCRs) are important targets for medicinal agents. Four different G protein families, G(s), G(i), G(q), and G(12), engage in their linkage to activation of receptor-specific signal transduction pathways. G(12) 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 G(12/13)-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 Galpha(12) and Galpha(13) 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 G(12/13)-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 G(12/13)-linked GPCRs can be quantified and therefore used for pharmacological studies of the pathway, and to discover active compounds in a G(12/13)-related disease context.

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.

G12/13-dependent signaling of G-protein-coupled receptors: disease context and impact on drug discovery.

G-protein-coupled receptors (GPCRs) transmit extracellular signals across the plasma membrane via intracellular activation of heterotrimeric G proteins. The signal transduction pathways of Gs, Gi and Gq protein families are widely studied, whereas signaling properties of G12 proteins are only emerging. Many GPCRs were found to couple to G12/13 proteins in addition to coupling to one or more other types of G proteins. G12/13 proteins couple GPCRs to activation of the small monomeric GTPase RhoA. Activation of RhoA modulates various downstream effector systems relevant to diseases such as hypertension, artherosclerosis, asthma and cancer. GPCR screening assays exist for Gs-, Gi- and Gq-linked pathways, whereas a drug-screening assay for the G12-Rho pathway was developed only recently. The review gives an overview of the present understanding of the G12/13-related biology of GPCRs.

Novel GPCR screening approach: indirect identification of S1P receptor agonists in antagonist screening using a calcium assay.

To elucidate the physiological function of sphingosine 1-phosphate receptors 1-3 (S1P1-3) we aimed to identify selective ligands for these GPCRs. S1P2 and S1P3 are coupled to Gq, and are, therefore, linked to the phospholipase C/IP3/calcium pathway. S1P1 is solely coupled to Gi and was artificially linked to calcium signaling by coexpression of Galpha 16. The three receptors desensitized on challenge of cells with an agonist (i.e., agonists appeared as antagonists in a second calcium measurement). We screened a compound library for inhibitors of S1P-stimulated calcium signals, and we could identify agonists and antagonists with a single measurement. Agonism and antagonism were confirmed by recording compound-and S1P-induced calcium signals from the same assay well. For the three receptors, we found a reciprocal correlation of agonism and "apparent" antagonism of agonists. In addition, agonists indirectly discovered by this approach do not promote calcium mobilization through endogenous GPCRs.

Pathways of transduction engaged by sphingosine 1-phosphate through G protein-coupled receptors.

Pathways of transduction employed by receptors for sphingosine 1-phosphate (S1P) are identified by the nature of second messengers and/or downstream targets regulated and, more formally, by direct assays of heterotrimeric G protein activation. The different methods generally agree. S1P1 couples to members of the Gi family, apparently selectively, although reported pertussis toxin (PTX)-insensitive actions make categorical statements regarding exclusivity difficult. S1P2 and S1P3 couple to members of the Gi, Gq, and G12/13 families. S1P4 couples to Gi and possibly G12/13, while S1P5 couples to Gi and G12/13 but not to Gq. In virtually all circumstances, coupling of S1P receptors to Gi is reflected in PTX-sensitive inhibition of adenylyl cyclase, activation of extracellular-regulated kinases (ERKs), and, depending on the cell, activation of phospholipase C (PLC). Coupling to Gq is reflected in PTX-insensitive activation of phospholipase C. Coupling to G12/13 is reflected in activation of Rho and subsequent activation of serum response factor (SRF). Specific linkages have been verified in almost all instances by receptor-promoted [35S]GTPgammaS/GDP exchange on identified G proteins.