Mark Wheatley

Lifting the lid on GPCRs: the role of extracellular loops

GPCRs exhibit a common architecture of seven transmembrane helices (TMs) linked by intracellular loops and extracellular loops (ECLs). Given their peripheral location to the site of G‐protein interaction, it might be assumed that ECL segments merely link the important TMs within the helical bundle of the receptor. However, compelling evidence has emerged in recent years revealing a critical role for ECLs in many fundamental aspects of GPCR function, which supported by recent GPCR crystal structures has provided mechanistic insights. This review will present current understanding of the key roles of ECLs in ligand binding, activation and regulation of both family A and family B GPCRs.

Lifting The Lid On G-Protein-Coupled Receptors: The Role Of Extracellular Loops.

GPCRs exhibit a common architecture of seven transmembrane helices (TMs) linked by intracellular loops and extracellular loops (ECLs). Given their peripheral location to the site of G‐protein interaction, it might be assumed that ECL segments merely link the important TMs within the helical bundle of the receptor. However, compelling evidence has emerged in recent years revealing a critical role for ECLs in many fundamental aspects of GPCR function, which supported by recent GPCR crystal structures has provided mechanistic insights. This review will present current understanding of the key roles of ECLs in ligand binding, activation and regulation of both family A and family B GPCRs.

Systematic analysis of the entire second extracellular loop of the V(1a) vasopressin receptor: key residues, conserved throughout a G-protein-coupled receptor family, identified.

The roles of extracellular residues of G-protein-coupled receptors (GPCRs) are not well defined compared with residues in transmembrane helices. Nevertheless, it has been established that extracellular domains of both peptide-GPCRs and amine-GPCRs incorporate functionally important residues. Extracellular loop 2 (ECL2) has attracted particular interest, because the x-ray structure of bovine rhodopsin revealed that ECL2 projects into the binding crevice within the transmembrane bundle. Our study provides the first comprehensive investigation into the role of the individual residues comprising the entire ECL2 domain of a small peptide-GPCR. Using the V1a vasopressin receptor, systematic substitution of all of the ECL2 residues by Ala generated 30 mutant receptors that were characterized pharmacologically. The majority of these mutant receptor constructs (24 in total) had essentially wild-type ligand binding and intracellular signaling characteristics, indicating that these residues are not critical for normal receptor function. However, four aromatic residues Phe189, Trp206, Phe209, and Tyr218 are important for agonist binding and receptor activation and are highly conserved throughout the neurohypophysial hormone subfamily of peptide-GPCRs. Located in the middle of ECL2, juxtaposed to the highly conserved disulfide bond, Trp206 and Phe209 project into the binding crevice. Indeed, Phe209 is part of the Cys-X-X-X-Ar (where Ar is an aromatic residue) motif, which is well conserved in both peptide-GPCRs and amine-GPCRs. In contrast, Phe189 and Tyr218, located at the extreme ends of ECL2, may be important for determining the position of the ECL2 cap over the binding crevice. This study provides mechanistic insight into the roles of highly conserved ECL2 residues.

Structural analysis of a nanoparticle containing a lipid bilayer used for detergent-free extraction of membrane proteins

In the past few years there has been a growth in the use of nanoparticles for stabilizing lipid membranes that contain embedded proteins. These bionanoparticles provide a solution to the challenging problem of membrane protein isolation by maintaining a lipid bilayer essential to protein integrity and activity. We have previously described the use of an amphipathic polymer (poly(styrene-co-maleic acid), SMA) to produce discoidal nanoparticles with a lipid bilayer core containing the embedded protein. However the structure of the nanoparticle itself has not yet been determined. This leaves a major gap in understanding how the SMA stabilizes the encapsulated bilayer and how the bilayer relates physically and structurally to an unencapsulated lipid bilayer. In this paper we address this issue by describing the structure of the SMA lipid particle (SMALP) using data from small angle neutron scattering (SANS), electron microscopy (EM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC) and nuclear magnetic resonance spectroscopy (NMR). We show that the particle is disc shaped containing a polymer “bracelet” encircling the lipid bilayer. The structure and orientation of the individual components within the bilayer and polymer are determined showing that styrene moieties within SMA intercalate between the lipid acyl chains. The dimensions of the encapsulated bilayer are also determined and match those measured for a natural membrane. Taken together, the description of the structure of the SMALP forms the foundation for future development and applications of SMALPs in membrane protein production and analysis.

Molecular pharmacology of V1a vasopressin receptors

1. Vasopressin, a mammalian neurohypophysial peptide hormone, has diverse physiological actions. 2. Pharmacological studies, using a range of mammalian tissues, have identified three subtypes of vasopressin receptor. 3. The V1a subtype of vasopressin receptor is widely distributed and mediates many central and peripheral actions of vasopressin. 4. The development of subtype-selective vasopressin analogues has provided valuable tools for pharmacological and physical studies of the V1a receptor protein. 5. Pharmacological differences indicate species heterogeneity in the characteristics of V1a receptors and in the expression of hepatic V1a receptors. 6. The cloning of neurohypophysial hormone receptor proteins allows structural and functional comparison of the V1a vasopressin receptors with other G-protein-coupled receptors.

G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR–SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR–SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR–SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([3H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.

Neuropeptides, Amines and Amino Acids as Mediators of the Sympathetic Effects of Paraventricular Nucleus Activation in the Rat

The aim of the present study was to determine the influence on renal sympathetic nerve activity of the different chemically coded neuronal phenotypes that project from the paraventricular nucleus (PVN) to the spinal cord. Experiments were carried out on male Wistar rats anaesthetised with chloralose and urethane. Changes in renal sympathetic nerve activity were measured following activation of neurones in the PVN with D,L‐homocysteic acid (100 nl, 200 mM), before and following intrathecal application of glutamate, vasopressin, oxytocin, dopamine and their receptor antagonists. Excitatory and inhibitory effects on renal sympathetic nerve activity were elicited by PVN stimulation. PVN excitatory effects were mimicked by intrathecal administration of glutamate and vasopressin and selectively antagonised by intrathecal administration of kynurenic acid and a V1a receptor antagonist, respectively. A low dose of dopamine increased renal sympathetic activity and this was selectively antagonised by haloperidol; however, the latter was without effect on PVN excitatory responses. A high dose of dopamine decreased renal sympathetic nerve activity and this was selectively blocked by a D1 dopamine receptor antagonist (SCH 23390), which also antagonised a minority of inhibitory responses obtained from the caudal extension of the PVN. Oxytocin also had two actions: in 5 rats it inhibited and in 10 rats it increased renal sympathetic nerve activity, both actions being blocked selectively by oxytocin receptor antagonists. Neither of the PVN effects on renal sympathetic nerve activity appeared to be dependent on oxytocin pathways. Tests with intrathecal administration of bicuculline showed that PVN inhibition of renal sympathetic nerve activity was not dependent on spinal GABAA receptor activation. The results show that PVN‐induced excitation of sympathetic activity to the kidney is mainly mediated by glutamate or vasopressin neurones whereas dopamine via D1 receptors may mediate some of the PVN inhibitory effects.

Charged extracellular residues, conserved throughout a G-protein-coupled receptor family, are required for ligand binding, receptor activation, and cell-surface expression.

For G-protein-coupled receptors (GPCRs) in general, the roles of extracellular residues are not well defined compared with residues in transmembrane helices (TMs). Nevertheless, extracellular residues are important for various functions in both peptide-GPCRs and amine-GPCRs. In this study, the V1a vasopressin receptor was used to systematically investigate the role of extracellular charged residues that are highly conserved throughout a subfamily of peptide-GPCRs, using a combination of mutagenesis and molecular modeling. Of the 13 conserved charged residues identified in the extracellular loops (ECLs), Arg116 (ECL1), Arg125 (top of TMIII), and Asp204 (ECL2) are important for agonist binding and/or receptor activation. Molecular modeling revealed that Arg125 (and Lys125) stabilizes TMIII by interacting with lipid head groups. Charge reversal (Asp125) caused re-ordering of the lipids, altered helical packing, and increased solvent penetration of the TM bundle. Interestingly, a negative charge is excluded at this locus in peptide-GPCRs, whereas a positive charge is excluded in amine-GPCRs. This contrasting conserved charge may reflect differences in GPCR binding modes between peptides and amines, with amines needing to access a binding site crevice within the receptor TM bundle, whereas the binding site of peptide-GPCRs includes more extracellular domains. A conserved negative charge at residue 204 (ECL2), juxtaposed to the highly conserved disulfide bond, was essential for agonist binding and signaling. Asp204 (and Glu204) establishes TMIII contacts required for maintaining the β-hairpin fold of ECL2, which if broken (Ala204 or Arg204) resulted in ECL2 unfolding and receptor dysfunction. This study provides mechanistic insight into the roles of conserved extracellular residues.

The third extracellular loop of G-protein-coupled receptors: more than just a linker between two important transmembrane helices

GPCRs (G-protein-coupled receptors) are a large family of structurally related proteins, which mediate their effects by coupling with G-proteins. Despite responding to a range of very diverse stimuli, these receptors exhibit a conserved tertiary structure comprising a bundle of seven TM (transmembrane) helices linked by alternating ECLs (extracellular loops) and ICLs (intracellular loops). The hydrophobic environment formed by the cluster of TM helices is functionally important. For example, the 11-cis retinal chromophore of rhodopsin forms a protonated Schiff base linkage to a lysine in TM7, deep within the helical bundle, and small ligands, such as amine neurotransmitters and non-peptide analogues of peptide hormones, also bind within the corresponding region of their cognate receptors. In addition, activation of GPCRs involves relative movement of TM helices to present G-protein interaction sites across the intracellular face of the receptor. Consequently, it might be assumed that the ECLs of the GPCR are inert peptide linkers that merely connect important TM helices. Focusing on ECL3 (third ECL), it is becoming increasingly apparent that this extracellular domain can fulfil a range of important roles with respect to GPCR signalling, including agonist binding, ligand selectivity and receptor activation.

Heterodimers and family-B GPCRs: RAMPs, CGRP and adrenomedullin

RAMPs (receptor activity-modifying proteins) are single-pass transmembrane proteins that associate with certain family-B GPCRs (G-protein-coupled receptors). Specifically for the CT (calcitonin) receptor-like receptor and the CT receptor, this results in profound changes in ligand binding and receptor pharmacology, allowing the generation of six distinct receptors with preferences for CGRP (CT gene-related peptide), adrenomedullin, amylin and CT. There are three RAMPs: RAMP1-RAMP3. The N-terminus appears to be the main determinant of receptor pharmacology, whereas the transmembrane domain contributes to association of the RAMP with the GPCR. The N-terminus of all members of the RAMP family probably contains two disulphide bonds; a potential third disulphide is found in RAMP1 and RAMP3. The N-terminus appears to be in close proximity to the ligand and plays a key role in its binding, either directly or indirectly. BIBN4096BS, a CGRP antagonist, targets RAMP1 and this gives the compound very high selectivity for the human CGRP(1) receptor.