Mutational and computational analysis of the alpha(1b)-adrenergic receptor. Involvement of basic and hydrophobic residues in receptor activation and G protein coupling.

To investigate their role in receptor coupling to G(q), we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the alpha(1b)-adrenergic receptor (AR). The wild type and mutated receptors were expressed in COS-7 cells and characterized for their ligand binding properties and ability to increase inositol phosphate accumulation. The experimental results have been interpreted in the context of both an ab initio model of the alpha(1b)-AR and of a new homology model built on the recently solved crystal structure of rhodopsin. Among the twenty-three basic amino acids mutated only mutations of three, Arg(254) and Lys(258) in the third intracellular loop and Lys(291) at the cytosolic extension of helix 6, markedly impaired the receptor-mediated inositol phosphate production. Additionally, mutations of two conserved hydrophobic residues, Val(147) and Leu(151) in the second intracellular loop had significant effects on receptor function. The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg(254), Lys(258), as well as Leu(151) are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein. In contrast, the residues belonging to the cytosolic extensions of helices 3 and 6 play a predominant role in the activation process of the alpha(1b)-AR. These findings contribute to the delineation of the molecular determinants of the alpha(1b)-AR/G(q) interface.

Molecular mechanisms of prostacyclin receptor signaling: The intracellular domains in G protein coupling

To better understand the mechanisms of how the human prostacyclin receptor (1P) mediates vasodilation and platelet anti-aggregation through Gs protein coupling, a strategy integrating multiple approaches including high resolution NMR experiments, synthetic peptide, fluorescence spectroscopy, molecular modeling, and recombinant protein was developed and used to characterize the structure/function relationship of important segments and residues of the IP receptor and the α-subunit of the Gs protein (Gαs). The first (iLP1) and third (iLP3) intracellular loops of the IP receptor, as well as the Gαs C-terminal domain, relevant to the Gs-mediated IP receptor signaling, were first identified by observation of the effects of the mini gene-expressed corresponding protein segments in HEK293 cells which co-expressed the receptor and Gαs. Evidence of the IP iLP1 domain interacted with the Gαs C-terminal domain was observed by fluorescence and NMR spectroscopic studies using a constrained synthetic peptide, which mimicked the IP iLP1 domain, and the synthetic peptide, which mimicked Gαs C-terminal domain. The solution structural models and the peptide-peptide interaction of the two synthetic protein segments were determined by high resolution NMR spectroscopy. The important residues in the corresponding domains of the IP receptor and the Gαs predicted by NMR chemical shift mapping were used to guide the identification of their protein-protein interaction in cells. A profile of the residues Arg42 - Ala48 of the IP iLP1 domain and the three residues Glu392 ∼ Leu394 of the Gαs C-terminal domain involved in the IP/Gs protein coupling were confirmed by recombinant proteins. The data revealed an intriguing speculation on the mechanisms of how the signal of the ligand-activated IP receptor is transmitted to the Gs protein in regulating vascular functions and homeostasis, and also provided substantial insights into other prostanoid receptor signaling. ^

Agonist-induced conformational changes in the G-protein-coupling domain of the β2 adrenergic receptor

The majority of extracellular physiologic signaling molecules act by stimulating GTP-binding protein (G-protein)-coupled receptors (GPCRs). To monitor directly the formation of the active state of a prototypical GPCR, we devised a method to site specifically attach fluorescein to an endogenous cysteine (Cys-265) at the cytoplasmic end of transmembrane 6 (TM6) of the β2 adrenergic receptor (β2AR), adjacent to the G-protein-coupling domain. We demonstrate that this tag reports agonist-induced conformational changes in the receptor, with agonists causing a decline in the fluorescence intensity of fluorescein-β2AR that is proportional to the biological efficacy of the agonist. We also find that agonists alter the interaction between the fluorescein at Cys-265 and fluorescence-quenching reagents localized to different molecular environments of the receptor. These observations are consistent with a rotation and/or tilting of TM6 on agonist activation. Our studies, when compared with studies of activation in rhodopsin, indicate a general mechanism for GPCR activation; however, a notable difference is the relatively slow kinetics of the conformational changes in the β2AR, which may reflect the different energetics of activation by diffusible ligands.

Receptor-G protein coupling is established by a potential conformational switch in the beta gamma complex.

Receptor-G protein interaction is characterized by cycles of association and dissociation. We present evidence which indicates that during receptor-G protein interaction, the C-terminal tail of the G protein gamma subunit, which is masked in the beta gamma complex, is exposed and establishes high-affinity contact with the receptor. This potential conformational switch provides a mechanism to regulate receptor-G protein coupling. This switch may also be significant for the role of the beta gamma complex in regulation of effector function.

The role of members of the pertussis toxin-sensitive family of G proteins in coupling receptors to the activation of the G protein-gated inwardly rectifying potassium channel

Inwardly rectifying potassium (K+) channels gated by G proteins (Kir3.x family) are widely distributed in neuronal, atrial, and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials, slowing the heart rate and modulating hormone release. They are directly activated by Gβγ subunits released from G protein heterotrimers of the Gi/o family upon appropriate receptor stimulation. Here we examine the role of isoforms of pertussis toxin (PTx)-sensitive G protein α subunits (Giα1–3 and GoαA) in mediating coupling between various receptor systems (A1, α2A, D2S, M4, GABAB1a+2, and GABAB1b+2) and the cloned counterpart of the neuronal channel (Kir3.1+3.2A). The expression of mutant PTx-resistant Gi/oα subunits in PTx-treated HEK293 cells stably expressing Kir3.1+3.2A allows us to selectively investigate that coupling. We find that, for those receptors (A1, α2A) known to interact with all isoforms, Giα1–3 and GoαA can all support a significant degree of coupling to Kir3.1+3.2A. The M4 receptor appears to preferentially couple to Giα2 while another group of receptors (D2S, GABAB1a+2, GABAB1b+2) activates the channel predominantly through Gβγ liberated from GoA heterotrimers. Interestingly, we have also found a distinct difference in G protein coupling between the two splice variants of GABAB1. Our data reveal selective pathways of receptor activation through different Gi/oα isoforms for stimulation of the G protein-gated inwardly rectifying K+ channel.

The influence of G protein subtype on agonist action at D-2 dopamine receptors

In previous studies, we have shown that agonists influence the ability of D-2 dopamine receptors to couple to G proteins and here we extend this work. The human D-2Short dopamine receptor and a natural polymorphism of this D-2Short(Ser(311)Cys), have been studied by co-expressing the receptors in insect cells with Gbeta(1)gamma(2) and either Galpha(o), Galpha(i1), Galpha(i2) or Galpha(i3) G protein subunits. These preparations have been used to study the G protein coupling profiles of the two receptors and the influence of agonists. Receptor/G protein coupling was analysed in dopamine/[H-3]spiperone competition binding experiments and through stimulation of [S-35]GTPgammaS binding. Although the Ser(311)Cys polymorphism itself had no appreciable effect on the G protein coupling specificity of the D-2 receptor, agonist stimulation of [S-35]GTPgammaS binding, revealed that both dopamine and (+)-3PPP showed a clear preference for Galpha(o) compared to the Galpha(i) subtypes, but quinpirole did not. These results indicate that agonists are able to stabilise different receptor conformations with different abilities to couple to G proteins. (C) 2004 Elsevier Ltd. All rights reserved.

Agonist binding, agonist affinity and agonist efficacy at G protein-coupled receptors

Measurements of affinity and efficacy are fundamental for work on agonists both in drug discovery and in basic studies on receptors. In this review I wish to consider methods for measuring affinity and efficacy at G protein coupled receptors (GPCRs). Agonist affinity may be estimated in terms of the dissociation constant for agonist binding to a receptor using ligand binding or functional assays. It has, however, been suggested that measurements of affinity are always contaminated by efficacy so that it is impossible to separate the two parameters. Here I show that for many GPCRs, if receptor/G protein coupling is suppressed, experimental measurements of agonist affinity using ligand binding (K-obs) provide quite accurate measures of the agonist microscopic dissociation constant (K-A). Also in pharmacological functional studies, good estimates of agonist dissociation constants are possible. Efficacy can be quantitated in several ways based on functional data ( maximal effect of the agonist (E-max), ratio of agonist dissociation constant to concentration of agonist giving half maximal effect in functional assay ( K-obs/ EC50), a combined parameter EmaxKobs/EC50). Here I show that EmaxKobs/EC50 provides the best assessment of efficacy for a range of agonists across the full range of efficacy for full to partial agonists. Considerable evidence now suggests that ligand efficacy may be dependent on the pathway used to assess it. The efficacy of a ligand may, therefore, be multidimensional. It is still, however, necessary to have accurate measures of efficacy in different pathways.

Specificity of somatostatin receptor and G -protein interactions as characterized by somatostatin receptor subtype specific antibodies

The neuropeptide somatostatin is a widely distributed general inhibitor of endocrine, exocrine, gastrointestinal and neural functions. The biological actions of somatostatin are initiated by interaction with high affinity, plasma membrane somatostatin receptors (sst receptors). Five sst receptor subtypes have been cloned and sequence analysis shows they are all members of the G protein coupled receptor superfamily. The G proteins play a pivotal role in sst receptor signal transduction and the specificity of somatostatin receptor-G protein coupling defines the possible range of cellular responses. However, the data for endogenous sst receptor and G protein coupling is very limited, and even when it is available, the sst receptor subtypes involved in G protein coupling and signal transduction are unknown due to the expression of multiple sst receptor subtypes in target cell lines or tissues of somatostatin.^ In an effort to characterize each individual sst receptor subtypes, antisera against unique C-terminal regions of different sst receptor subtypes have been developed in our lab. In this report, antisera made against the sst1, sst2A and sst4 receptors are characterized. They are highly specific to their corresponding receptors and efficiently immunoprecipitate the sst receptors. Using these antibodies, the cell lines expressing these sst receptor subtypes were identified with both immunoprecipitation and Western blot methods. The development of sst receptor subtype specific antibodies make it possible to determine the specificity of the sst receptor subtype and G protein coupling in target cells or tissues expressing multiple sst receptors, two questions were addressed by this thesis: (1) whether different cellular environments affect receptor subtype and G protein coupling; (2) whether different sst receptors couple to different G proteins in similar cellular environments.^ Taken together our findings, both sst1 and sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells, G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in GH$\sb4$C$\sb1$ cells. Further, sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in AR4-2J cells while sst4 receptors couple with G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells. Therefore, the G protein coupling of the same sst receptors in different cell lines is basically similar in that they all couple with multiple $\alpha$-subunits of the G$\rm \sb{i}$ proteins, suggesting cellular environment has little effect on receptor and G protein coupling. Moreover, different sst receptors have similar G protein coupling specificities in the same cell line, suggesting components other than receptor and G$\alpha$ subunits in the signal transduction pathways may contribute to specific functions of each sst receptor subtype. This series of experiments represent a novel approach in dissecting signal transduction pathways and may have general application in the field. Furthermore, this is the first systematic study of sst receptor subtype and G protein $\alpha$-subunit interaction in both transfected cells and in normal cell lines. The information generated will be very useful in our understanding of sst receptor signal transduction pathways and in directing future sst receptor research. ^

Mechanisms Governing the Activation and Trafficking of Yeast G Protein-coupled Receptors

We have addressed the mechanisms governing the activation and trafficking of G protein-coupled receptors (GPCRs) by analyzing constitutively active mating pheromone receptors (Ste2p and Ste3p) of the yeast Saccharomyces cerevisiae. Substitution of the highly conserved proline residue in transmembrane segment VI of these receptors causes constitutive signaling. This proline residue may facilitate folding of GPCRs into native, inactive conformations, and/or mediate agonist-induced structural changes leading to G protein activation. Constitutive signaling by mutant receptors is suppressed upon coexpression with wild-type, but not G protein coupling-defective, receptors. Wild-type receptors may therefore sequester a limiting pool of G proteins; this apparent “precoupling” of receptors and G proteins could facilitate signal production at sites where cell surface projections form during mating partner discrimination. Finally, rather than being expressed mainly at the cell surface, constitutively active pheromone receptors accumulate in post-endoplasmic reticulum compartments. This is in contrast to other defective membrane proteins, which apparently are targeted by default to the vacuole. We suggest that the quality-control mechanism that retains receptors in post-endoplasmic reticulum compartments may normally allow wild-type receptors to fold into their native, fully inactive conformations before reaching the cell surface. This may ensure that receptors do not trigger a response in the absence of agonist.

Investigating G protein signalling bias at the glucagon-like peptide-1 receptor in yeast

Background and Purpose The glucagon-like peptide 1 (GLP-1) receptor performs an important role in glycaemic control, stimulating the release of insulin. It is an attractive target for treating type 2 diabetes. Recently, several reports of adverse side effects following prolonged use of GLP-1 receptor therapies have emerged: most likely due to an incomplete understanding of signalling complexities. Experimental Approach We describe the expression of the GLP-1 receptor in a panel of modified yeast strains that couple receptor activation to cell growth via single Gα/yeast chimeras. This assay enables the study of individual ligand-receptor G protein coupling preferences and the quantification of the effect of GLP-1 receptor ligands on G protein selectivity. Key Results The GLP-1 receptor functionally coupled to the chimeras representing the human Gαs, Gαi and Gαq subunits. Calculation of the dissociation constant for a receptor antagonist, exendin-3 revealed no significant difference between the two systems. We obtained previously unobserved differences in G protein signalling bias for clinically relevant therapeutic agents, liraglutide and exenatide; the latter displaying significant bias for the Gαi pathway. We extended the use of the system to investigate small-molecule allosteric compounds and the closely related glucagon receptor. Conclusions and Implications These results provide a better understanding of the molecular events involved in GLP-1 receptor pleiotropic signalling and establish the yeast platform as a robust tool to screen for more selective, efficacious compounds acting at this important class of receptors in the future. © 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.

Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins

Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine.

Functional coupling of the human dopamine D-2 receptor with G alpha(i1), G alpha(i2), G alpha(i3) and G alpha(o) G proteins: evidence for agonist regulation of G protein selectivity

1 The human dopamine D-2long (D-2L) receptor was expressed with four different G proteins in Sf9 cells using the baculovirus expression system. When co-expressed with G(i)/G(o) G proteins (G(i1)alpha, G(i2)alpha, G(i3)alpha, or G(o)alpha, plus Gbeta(1) and Ggamma(2)) the receptor displayed a high-affinity binding site for the agonists (dopamine and NPA), which was sensitive to GTP (100 mum), demonstrating interaction between the receptor and the different G proteins. 2 The receptor to G protein ratio (R: G ratio) was evaluated using [H-3]-spiperone saturation binding (R) and [S-35]-GTPgammaS saturation binding (G). R: G ratios of 1: 12, 1: 3, 1: 14 and 1: 5 were found for G(i1), G(i2), G(i3), and Go preparations, respectively. However, when R:G ratios of 1:2 and 1: 12 were compared for G(i2) and G(o), no difference was found for the stimulation of [S-35]-GTPgammaS binding. 3 Several agonists were tested for their ability to stimulate [S-35]-GTPgammaS binding to membranes co-expressing the receptor and various G proteins. All the compounds tested showed agonist activity in preparations expressing G(i3) and G(o). However, for G(i2) and G(i1) preparations, compounds such as S-(-)-3-PPP and p-tyramine were unable to stimulate [S-35]-GTPyS binding. 4 Most of the compounds showed higher relative efficacies (compared to dopamine) and higher potencies in the preparation expressing G(o). Comparison of the effects of different agonists in the different preparations showed that each agonist differentially activates the four G proteins. 5 We conclude that the degree of selectivity of G protein activation by the D-2L receptor can depend on the conformation of the receptor stabilised by an agonist.

Small molecular probes for G-protein-coupled C5a receptors: Conformationally constrained antagonists derived from the C terminus of the human plasma protein C5a

Activation of the human complement system of plasma proteins in response to infection or injury produces a 4-helix bundle glycoprotein (74 amino acids) known as C5a. C5a binds to G-protein-coupled receptors on cell surfaces triggering receptor-ligand internalization, signal transduction, and powerful inflammatory responses. Since excessive levels of C5a are associated with autoimmune and chronic inflammatory disorders, inhibitors of receptor activation may have therapeutic potential. We now report solution structures and receptor-binding and antagonist activities for some of the first small molecule antagonists of C5a derived from its hexapeptide C terminus. The antagonist NMe-Phe-Lys-Pro-D-Cha-Trp-D-Arg-CO2H (1) surprisingly shows an unusually well-defined solution structure as determined by H-1 NMR spectroscopy. This is one of the smallest acyclic peptides found to possess a defined solution conformation, which can be explained by the constraining role of intramolecular hydrogen bonding. NOE and coupling constant data, slow deuterium exchange, and a low dependence on temperature for the chemical shift of the D-Cha-NH strongly indicate an inverse gamma turn stabilized by a D-Cha-NH ... OC-Lys hydrogen bond. Smaller conformational populations are associated with a hydrogen bond between Trp-NH ... OC-Lys, defining a type II beta turn distorted by the inverse gamma turn incorporated within it. An excellent correlation between receptor-affinity and antagonist activity is indicated for a limited set of synthetic peptides. Conversion of the C-terminal carboxylate of 1 to an amide decreases antagonist potency 5-fold, but potency is increased up to 10-fold over 1 if the amide bond is made between the C-terminal carboxylate and a Lys/Orn side chain to form a cyclic analogue. The solution structure of cycle 6 also shows gamma and beta turns; however, the latter occurs in a different position, and there are clear conformational changes in 6 vs 1 that result in enhanced activity. These results indicate that potent C5a antagonists can be developed by targeting site 2 alone of the C5a receptor and define a novel pharmacophore for developing powerful receptor probes or drug candidates.

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system.

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.

Protease-activated receptor 2 (PAR2) protein and transient receptor potential vanilloid 4 (TRPV4) protein coupling is required for sustained inflammatory signaling

G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR(2)), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR(2) regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR(2) agonist stimulated a transient increase in [Ca(2+)](i). TRPV4 expression led to a markedly sustained increase in [Ca(2+)](i). Removal of extracellular Ca(2+) and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A(2) and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR(2) generates arachidonic acid-derived lipid mediators, such as 5',6'-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR(2)-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR(2) was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR(2) signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR(2)-stimulated neurogenic inflammation. Thus, PAR(2) activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR(2).