GPCRs in the round: SMA-like copolymers and SMALPs as a platform for investigating GPCRs.

G-protein-coupled receptors (GPCRs) are the largest family of membrane proteins, regulate a plethora of physiological responses and are the therapeutic target for 30-40% of clinically-prescribed drugs. They are integral membrane proteins deeply embedded in the plasma membrane where they activate intracellular signalling via coupling to G-proteins and ß-arrestin. GPCRs are in intimate association with the bilayer lipids and that lipid environment regulates the signalling functions of GPCRs. This complex lipid 'landscape' is both heterogeneous and dynamic. GPCR function is modulated by bulk membrane properties including membrane fluidity, microdomains, curvature, thickness and asymmetry but GPCRs are also regulated by specific lipid:GPCR binding, including cholesterol and anionic lipids. Understanding the molecular mechanisms whereby GPCR signalling is regulated by lipids is a very active area of research currently. A major advance in membrane protein research in recent years was the application of poly(styrene-co-maleic acid) (SMA) copolymers. These spontaneously generate SMA lipid particles (SMALPs) encapsulating membrane protein in a nano-scale disc of cell membrane, thereby removing the historical need for detergent and preserving lipid:GPCR interaction. The focus of this review is how GPCR-SMALPs are increasing our understanding of GPCR structure and function at the molecular level. Furthermore, an increasing number of 'second generation' SMA-like copolymers have been reported recently. These are reviewed from the context of increasing our understanding of GPCR molecular mechanisms. Moreover, their potential as a novel platform for downstream biophysical and structural analyses is assessed and looking ahead, the translational application of SMA-like copolymers to GPCR drug discovery programmes in the future is considered.

Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor.

Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a ß-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.

Expression and purification of recombinant G protein-coupled receptors: A review.

Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of their advantages and disadvantages, as well as examples of receptors successfully expressed in these systems. Additionally, an overview is given on the use of detergents and the styrene maleic acid (SMA) co-polymer for membrane solubilisation, as well as purification techniques.

Microbial expression systems for membrane proteins.

Despite many high-profile successes, recombinant membrane protein production remains a technical challenge; it is still the case that many fewer membrane protein structures have been published than those of soluble proteins. However, progress is being made because empirical methods have been developed to produce the required quantity and quality of these challenging targets. This review focuses on the microbial expression systems that are a key source of recombinant prokaryotic and eukaryotic membrane proteins for structural studies. We provide an overview of the host strains, tags and promoters that, in our experience, are most likely to yield protein suitable for structural and functional characterization. We also catalogue the detergents used for solubilization and crystallization studies of these proteins. Here, we emphasize a combination of practical methods, not necessarily high-throughput, which can be implemented in any laboratory equipped for recombinant DNA technology and microbial cell culture.

Photoaffinity Cross-Linking and Unnatural Amino Acid Mutagenesis Reveal Insights into Calcitonin Gene-Related Peptide Binding to the Calcitonin Receptor-like Receptor/Receptor Activity-Modifying Protein 1 (CLR/RAMP1) Complex.

Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1). How CGRP interacts with the transmembrane domain (including the extracellular loops) of this family B receptor remains unclear. In this study, a photoaffinity cross-linker, p-azido l-phenylalanine (azF), was incorporated into CLR, chiefly in the second extracellular loop (ECL2) using genetic code expansion and unnatural amino acid mutagenesis. The method was optimized to ensure efficient photolysis of azF residues near the transmembrane bundle of the receptor. A CGRP analogue modified with fluorescein at position 15 was used for detection of ultraviolet-induced cross-linking. The methodology was verified by confirming the known contacts of CGRP to the extracellular domain of CLR. Within ECL2, the chief contacts were I284 on the loop itself and L291, at the top of the fifth transmembrane helix (TM5). Minor contacts were noted along the lip of ECL2 between S286 and L290 and also with M223 in TM3 and F349 in TM6. Full length molecular models of the bound receptor complex suggest that CGRP sits at the top of the TM bundle, with Thr6 of the peptide making contacts with L291 and H295. I284 is likely to contact Leu12 and Ala13 of CGRP, and Leu16 of CGRP is at the ECL/extracellular domain boundary of CLR. The reduced potency, Emax, and affinity of [Leu16Ala]-human α CGRP are consistent with this model. Contacts between Thr6 of CGRP and H295 may be particularly important for receptor activation.

Relative Antagonism of Mutants of the CGRP Receptor Extracellular Loop 2 Domain (ECL2) Using a Truncated Competitive Antagonist (CGRP8-37): Evidence for the Dual Involvement of ECL2 in the Two-Domain Binding Model.

The second extracellular loop (ECL2) of the G protein-coupled receptor (GPCR) family is important for ligand interaction and drug discovery. ECL2 of the family B cardioprotective calcitonin gene-related peptide (CGRP) receptor is required for cell signaling. Family B GPCR ligands have two regions; the N-terminus mediates receptor activation, and the remainder confers high-affinity binding. Comparing antagonism of CGRP8-37 at a number of point mutations of ECL2 of the CGRP receptor, we show that the ECL2 potentially facilitates interaction with up to the 18 N-terminal residues of CGRP. This has implications for understanding family B GPCR activation and for drug design at the CGRP receptor.

Receptor Activity-modifying Protein-directed G Protein Signaling Specificity for the Calcitonin Gene-related Peptide Family of Receptors.

The calcitonin gene-related peptide (CGRP) family of G protein-coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in a Gαs-mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gαs and Gαq but also identify a Gαi component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMP-dependent signaling bias among the Gαs, Gαi, and Gαq/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology.

Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties.

Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.

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.

GPCR-styrene maleic acid lipid particles (GPCR-SMALPs): their nature and potential.

G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a 'molecular pastry cutter' to 'solubilize' GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR-SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR.

Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks.

BACKGROUND: Pichia pastoris is a widely-used host for recombinant protein production; expression is typically driven by methanol-inducible alcohol oxidase (AOX) promoters. Recently this system has become an important source of recombinant G protein-coupled receptors (GPCRs) for structural biology and drug discovery. The influence of diverse culture parameters (such as pH, dissolved oxygen concentration, medium composition, antifoam concentration and culture temperature) on productivity has been investigated for a wide range of recombinant proteins in P. pastoris. In contrast, the impact of the pre-induction phases on yield has not been as closely studied. In this study, we examined the pre-induction phases of P. pastoris bioreactor cultivations producing three different recombinant proteins: the GPCR, human A(2a) adenosine receptor (hA(2a)R), green fluorescent protein (GFP) and human calcitonin gene-related peptide receptor component protein (as a GFP fusion protein; hCGRP-RCP-GFP). RESULTS: Functional hA(2a)R was detected in the pre-induction phases of a 1 L bioreactor cultivation of glycerol-grown P. pastoris. In a separate experiment, a glycerol-grown P. pastoris strain secreted soluble GFP prior to methanol addition. When glucose, which has been shown to repress AOX expression, was the pre-induction carbon source, hA(2a)R and GFP were still produced in the pre-induction phases. Both hA(2a)R and GFP were also produced in methanol-free cultivations; functional protein yields were maintained or increased after depletion of the carbon source. Analysis of the pre-induction phases of 10 L pilot scale cultivations also demonstrated that pre-induction yields were at least maintained after methanol induction, even in the presence of cytotoxic concentrations of methanol. Additional bioreactor data for hCGRP-RCP-GFP and shake-flask data for GFP, horseradish peroxidase (HRP), the human tetraspanins hCD81 and CD82, and the tight-junction protein human claudin-1, demonstrated that bioreactor but not shake-flask cultivations exhibit recombinant protein production in the pre-induction phases of P. pastoris cultures. CONCLUSIONS: The production of recombinant hA(2a)R, GFP and hCGRP-RCP-GFP can be detected in bioreactor cultivations prior to methanol induction, while this is not the case for shake-flask cultivations of GFP, HRP, hCD81, hCD82 and human claudin-1. This confirms earlier suggestions of leaky expression from AOX promoters, which we report here for both glycerol- and glucose-grown cells in bioreactor cultivations. These findings suggest that the productivity of AOX-dependent bioprocesses is not solely dependent on induction by methanol. We conclude that in order to maximize total yields, pre-induction phase cultivation conditions should be optimized, and that increased specific productivity may result in decreased biomass yields.

Intracellular pathways of calcitonin gene-related peptide-induced relaxation of human coronary arteries: A key role for Gßγ subunit instead of cAMP.

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP) is a potent vasodilator. While its signalling is assumed to be mediated via increases in cAMP, this study focused on elucidating the actual intracellular signalling pathways involved in CGRP-induced relaxation of human isolated coronary arteries (HCA). EXPERIMENTAL APPROACH: HCA were obtained from heart valve donors (27 M, 25 F, age 54 ± 2 years). Concentration-response curves to human α-CGRP or forskolin were constructed in HCA segments, incubated with different inhibitors of intracellular signalling pathways, and intracellular cAMP levels were measured with and without stimulation. RESULTS: Adenylyl cyclase (AC) inhibitors SQ22536 + DDA and MDL-12330A, and PKA inhibitors Rp-8-Br-cAMPs and H89, did not inhibit CGRP-induced relaxation of HCA, nor did the guanylyl cyclase inhibitor ODQ, PKG inhibitor KT5823, EPAC1/2 inhibitor ESI09, potassium channel blockers TRAM-34 + apamin, iberiotoxin or glibenclamide, or the Gαq inhibitor YM-254890. Phosphodiesterase inhibitors induced a concentration-dependent decrease in the response to KCl but did not potentiate relaxation to CGRP. Relaxation to forskolin was not blocked by PKA or AC inhibitors, although AC inhibitors significantly inhibited the increase in cAMP. Inhibition of Gßγ subunits using gallein significantly inhibited the relaxation to CGRP in human coronary arteries. CONCLUSION: While CGRP signalling is generally assumed to act via cAMP, the CGRP-induced vasodilation in HCA was not inhibited by targeting this intracellular signalling pathway at different levels. Instead, inhibition of Gßγ subunits did inhibit the relaxation to CGRP, suggesting a different mechanism of CGRP-induced relaxation than generally believed.

G-protein-coupled receptors: from structural insights to functional mechanisms.

The papers resulting from the recent Biochemical Society Focused Meeting 'G-Protein-Coupled Receptors: from Structural Insights to Functional Mechanisms' held in Prato in September 2012 are introduced in the present overview. A number of future goals for GPCR (G-protein-coupled receptor) research are considered, including the need to develop biophysical and computational methods to explore the full range of GPCR conformations and their dynamics, the need to develop methods to take this into account for drug discovery and the importance of relating observations on isolated receptors or receptors expressed in model systems to receptor function in vivo.

The activation of the CGRP receptor.

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. CLR is an example of a family B GPCR. Unlike family A GPCRs, little is known about how these receptors are activated by their endogenous ligands. This review considers what is known about the activation of family B GPCRs and then considers how this might be applied to CLR, particularly in light of new knowledge of the crystal structures of family A GPCRs.

Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function.

The first and third extracellular loops (ECL) of G protein-coupled receptors (GPCRs) have been implicated in ligand binding and receptor function. This study describes the results of an alanine/leucine scan of ECLs 1 and 3 and loop-associated transmembrane (TM) domains of the secretin-like GPCR calcitonin receptor-like receptor which associates with receptor activity modifying protein 1 to form the CGRP receptor. Leu195Ala, Val198Ala and Ala199Leu at the top of TM2 all reduced αCGRP-mediated cAMP production and internalization; Leu195Ala and Ala199Leu also reduced αCGRP binding. These residues form a hydrophobic cluster within an area defined as the "minor groove" of rhodopsin-like GPCRs. Within ECL1, Ala203Leu and Ala206Leu influenced the ability of αCGRP to stimulate adenylate cyclase. In TM3, His219Ala, Leu220Ala and Leu222Ala have influences on αCGRP binding and cAMP production; they are likely to indirectly influence the binding site for αCGRP as well as having an involvement in signal transduction. On the exofacial surfaces of TMs 6 and 7, a number of residues were identified that reduced cell surface receptor expression, most noticeably Leu351Ala and Glu357Ala in TM6. The residues may contribute to the RAMP1 binding interface. Ile360Ala impaired αCGRP-mediated cAMP production. Ile360 is predicted to be located close to ECL2 and may facilitate receptor activation. Identification of several crucial functional loci gives further insight into the activation mechanism of this complex receptor system and may aid rational drug design.

The role of the extracellular loops of the CGRP receptor, a family B GPCR.

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity-modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. There have been few systematic studies of the ECLs (extracellular loops) of family B GPCRs. However, they are likely to be especially important for the interaction of the N-termini of the peptide agonists that are the natural agonists for these receptors. We have carried out alanine scans on all three ECLs of CLR, as well as their associated juxtamembrane regions. Residues within all three loops influence CGRP binding and receptor activation. Mutation of Ala203 and Ala206 on ECL1 to leucine increased the affinity of CGRP. Residues at the top of TM (transmembrane) helices 2 and 3 influenced CGRP binding and receptor activation. L351A and E357A in TM6/ECL3 reduced receptor expression and may be needed for CLR association with RAMP1. ECL2 seems especially important for CLR function; of the 16 residues so far examined in this loop, eight residues reduce the potency of CGRP at stimulating cAMP production when mutated to alanine.

RAMPs and CGRP receptors.

Receptor activity modifying protein 1 (RAMP1) forms a complex with calcitonin receptor-like receptor (CLR) to produce the receptor for calcitonin gene-related peptide (CGRP). RAMP1 has two main roles. It facilitates the cell-surface expression of CLR. It is also essential for the binding of CGRP to the receptor. It seems likely that Y66, F93, H97 and F101, amongst other residues, form a binding site for CLR. These cluster together on the same face of the extracellular portion of RAMP1, probably close to where it enters the plasma membrane. Residues at the other end of RAMP1 are most likely to be involved in CGRP recognition, although it is currently unclear how they do this. Within this area, W74 is important for the binding of the nonpeptide antagonist, BIBN4096BS, although it does not seem to be involved in the binding of CGRP itself. It has been shown that there is an epitope within residues 23-60 of CLR that are essential for RAMP recognition. Under some circumstances, changes in the expression of RAMP1 can alter the sensitivity of cells to CGRP, demonstrating that regulation of its levels may be of physiological or pathophysiological importance.

RAMPs as drug targets.

The receptor activity-modifying protein (RAMP) family of membrane proteins regulates G protein-coupled receptor (GPCR) function in several ways. RAMPs can alter their pharmacology and signalling as well as the trafficking of these receptors to and from the cell surface. Accordingly, RAMPs may be exploited as drug targets, offering new opportunities for regulating the function of therapeutically relevant RAMP-interacting GPCRs. For example, several small molecule antagonists of RAMP1/ calcitonin receptor-like receptor complexes, which block the actions of the neuropeptide calcitonin gene-related peptide are in development for the treatment of migraine headache.

Determining the Effects of Differential Expression of GRKs and ß-arrestins on CLR-RAMP Agonist Bias.

Signalling of the calcitonin-like receptor (CLR) is multifaceted, due to its interaction with receptor activity modifying proteins (RAMPs), and three endogenous peptide agonists. Previous studies have focused on the bias of G protein signalling mediated by the receptor and receptor internalisation of the CLR-RAMP complex has been assumed to follow the same pattern as other Class B1 G Protein-Coupled Receptors (GPCRs). Here we sought to measure desensitisation of the three CLR-RAMP complexes in response to the three peptide agonists, through the measurement of ß-arrestin recruitment and internalisation. We then delved further into the mechanism of desensitisation through modulation of ß-arrestin activity and the expression of GPCR kinases (GRKs), a key component of homologous GPCR desensitisation. First, we have shown that CLR-RAMP1 is capable of potently recruiting ß-arrestin1 and 2, subsequently undergoing rapid endocytosis, and that CLR-RAMP2 and -RAMP3 also utilise these pathways, although to a lesser extent. Following this we have shown that agonist-dependent internalisation of CLR is ß-arrestin dependent, but not required for full agonism. Overexpression of GRK2-6 was then found to decrease receptor signalling, due to an agonist-independent reduction in surface expression of the CLR-RAMP complex. These results represent the first systematic analysis of the importance of ß-arrestins and GRKs in CLR-RAMP signal transduction and pave the way for further investigation regarding other Class B1 GPCRs.