Structure-function analysis of RAMP1 by Alanine Mutagenesis

Receptor activity modifying protein 1 (RAMP1) is an integral component of several receptors including the calcitonin gene-related peptide (CGRP) receptor. It forms a complex with the calcitonin receptor-like receptor (CLR) and is required for receptor trafficking and ligand binding. The N-terminus of RAMP1 comprises three helices. The current study investigated regions of RAMP1 important for CGRP or CLR interactions by alanine mutagenesis. Modeling suggested the second and third helices were important in protein-protein interactions. Most of the conserved residues in the N-terminus (M48, W56, Y66, P85, N66, H97, F101, D113, P114, P115), together with a further 13 residues spread throughout three helices of RAMP1, were mutated to alanine and coexpressed with CLR in Cos 7 cells. None of the mutations significantly reduced RAMP expression. Of the nine mutants from helix 1, only M48A had any effect, producing a modest reduction in trafficking of CLR to the cell surface. In helix 2 Y66A almost completely abolished CLR trafficking; L69A and T73A reduced the potency of CGRP to produce cAMP. In helix 3, H97A abolished CLR trafficking; P85A, N86A, and F101A had caused modest reductions in CLR trafficking and also reduced the potency of CGRP on cAMP production. F93A caused a modest reduction in CLR trafficking alone and L94A increased cAMP production. The data are consistent with a CLR recognition site particularly involving Y66 and H97, with lesser roles for adjacent residues in helix 3. L69 and T73 may contribute to a CGRP recognition site in helix 2 also involving nearby residues.

Ligand binding and activation of the CGRP receptor

The receptor for CGRP (calcitonin gene-related peptide) is a heterodimer between a GPCR (G-protein-coupled receptor), CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity-modifying protein 1). Models have been produced of RAMP1 and CLR. It is likely that the C-terminus of CGRP interacts with the extracellular N-termini of CLR and RAMP1; the extreme N-terminus of CLR is particularly important and may interact directly with CGRP and also with RAMP1. The N-terminus of CGRP interacts with the TM (transmembrane) portion of the receptor; the second ECL (extracellular loop) is especially important. Receptor activation is likely to involve the relative movements of TMs 3 and 6 to create a G-protein-binding pocket, as in Family A GPCRs. Pro321 in TM6 appears to act as a pivot. At the base of TMs 2 and 3, Arg151, His155 and Glu211 may form a loose equivalent of the Family A DRY (Asp-Arg-Tyr) motif. Although the details of this proposed activation mechanism clearly do not apply to all Family B GPCRs, the broad outlines may be conserved. ©The Authors.

Role of β3-adrenergic receptors in the action of a tumour lipid mobilizing factor

Induction of lipolysis in murine white adipocytes, and stimulation of adenylate cyclase in adipocyte plasma membranes, by a tumour-produced lipid mobilizing factor, was attenuated by low concentrations (10-7-10-5M) of the specific β3-adrenoceptor antagonist SR59230A. Lipid mobilizing factor (250 nM) produced comparable increases in intracellular cyclic AMP in CHOKI cells transfected with the human β3-adrenoceptor to that obtained with isoprenaline (1 nM). In both cases cyclic AMP production was attenuated by SR59230A confirming that the effect is mediated through a β3-adrenoceptor. A non-linear regression analysis of binding of lipid mobilizing factor to the β3-adrenoceptor showed a high affinity binding site with a Kd value 78±45 nM and a Bmax value (282±1 fmole mg protein-1) comparable with that of other β3-adrenoceptor agonists. These results suggest that lipid mobilizing factor induces lipolysis through binding to a β3-adrenoceptor. © 2002 The Cancer Research Campaign.

The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network

Early endosome-to-trans-Golgi network (TGN) transport is organized by the retromer complex. Consisting of cargo-selective and membrane-bound subcomplexes, retromer coordinates sorting with membrane deformation and carrier formation. Here, we describe four mammalian retromers whose membrane-bound subcomplexes contain specific combinations of the sorting nexins (SNX), SNX1, SNX2, SNX5, and SNX6. We establish that retromer requires a dynamic spatial organization of the endosomal network, which is regulated through association of SNX5/SNX6 with the p150(glued) component of dynactin, an activator of the minus-end directed microtubule motor dynein; an association further defined through genetic studies in C. elegans. Finally, we also establish that the spatial organization of the retromer pathway is mediated through the association of SNX1 with the proposed TGN-localized tether Rab6-interacting protein-1. These interactions describe fundamental steps in retromer-mediated transport and establish that the spatial organization of the retromer network is a critical element required for efficient retromer-mediated sorting.

Structure-function analysis of RAMP1-RAMP3 chimeras

The role of receptor activity modifying protein 1 (RAMP1) in forming receptors with the calcitonin receptor-like receptor (CLR) and the calcitonin receptor (CTR) was examined by producing chimeras between RAMP1 and RAMP3. RAMPs have three extracellular helices. Exchange of helix 1 of the RAMPs or residues 62-69 in helix 2 greatly reduced CLR trafficking (a marker for CLR association). Modeling suggests that these exchanges alter the CLR recognition site on RAMP1, which is more exposed than on RAMP3. Exchange of residues 86-89 of RAMP1 had no effect on the trafficking of CLR but reduced the potency of human (h) alphaCGRP and adrenomedullin. However, these alterations to RAMP1 had no effect on the potency of hbetaCGRP. These residues of RAMP1 lie at the junction of helix 3 and its connecting loop with helix 2. Modeling suggests that the loop is more exposed in RAMP1 than RAMP3; it may play an important role in peptide binding, either directly or indirectly. Exchange of residues 90-94 of RAMP1 caused a modest reduction in CLR expression and a 15-fold decrease in CGRP potency. It is unlikely that the decrease in expression is enough to explain the reduction in potency, and so these may have dual roles in recognizing CLR and CGRP. For CTR, only 6 out of 26 chimeras covering the extracellular part of RAMP1 did not reduce agonist potency. Thus the association of CTR with RAMP1 seems more sensitive to changes in RAMP1 structure induced by the chimeras than is CLR.

Transglutaminase 2 is involved in autophagosome maturation

Autophagy is a highly conserved cellular process responsible for the degradation of long-lived proteins and organelles. Autophagy occurs at low levels under normal conditions, but it is enhanced in response to stress, e.g. nutrient deprivation, hypoxia, mitochondrial dysfunction and infection. "Tissue" transglutaminase (TG2) accumulates, both in vivo and in vitro, to high levels in cells under stressful conditions. Therefore, in this study, we investigated whether TG2 could also play a role in the autophagic process. To this end, we used TG2 knockout mice and cell lines in which the enzyme was either absent or overexpressed. The ablation of TG2 protein both in vivo and in vitro, resulted in an evident accumulation of microtubule-associated protein 1 light chain 3 cleaved isoform II (LC3 II) on pre-autophagic vesicles, suggesting a marked induction of autophagy. By contrast, the formation of the acidic vesicular organelles in the same cells was very limited, indicating an impairment of the final maturation of autophagolysosomes. In fact, the treatment of TG2 proficient cells with NH4Cl, to inhibit lysosomal activity, led to a marked accumulation of LC3 II and damaged mitochondria similar to what we observed in TG2-deficient cells. These data indicate a role for TG2-mediated post-translational modifications of proteins in the maturation of autophagosomes accompanied by the accumulation of many damaged mitochondria.

The mode of action of a lipid mobilising factor in cancer cachexia

Cachexia is characterised by a progressive weight loss due to depletion of both skeletal muscle and adipose tissue. The loss of adipose tissue is due to the production of a tumour-derived lipid mobilising factor (LMF), which has been shown to directly induce lipolysis in isolated epididymal murine white adipocytes. The administration of LMF to a non-tumour bearing mice produced a rapid weight loss, with a specific reduction in carcass lipid with also some redistribution of lipid with the accumulation of lipid in the liver. There was also up-regulation of uncoupling protein-1 and -2 mRNA and protein expression in brown adipose tissue, suggesting that an adaptive process occurs due to increased energy mobilisation. There was also up-regulation of UCP-2 in the livers of LMF treated mice, suggesting a protective mechanism to the build up of lipid in the livers, which would produce free radical by-products. LMF was also shown to stimulate cyclic AMP production in CHO-K1 cells transfected with human -3 adrenergic receptors and inhibited by the -β3 antagonist SR59230A. LMF binding was also inhibited by SR59230A in isolated receptors. This suggests that LMF mediates its effects through a β3 adrenergic receptor. There were also changes in glucose and fatty acid uptake in LMF treated mice, which suggests metabolic changes are occurring. The study suggests that a tumour derived lipolytic factor acts through the 3 adrenoceptor producing effects on lipid mobilisation, energy expenditure and glucose metabolism.

Immuno-potentiating activity of surfactant vesicles in DNA and subunit vaccination

Enhanced immune responses for DNA and subunit vaccines potentiated by surfactant vesicle based delivery systems outlined in the present study, provides proof of principle for the beneficial aspects of vesicle mediated vaccination. The dehydration-rehydration technique was used to entrap plasmid DNA or subunit antigens into lipid-based (liposomes) or non-ionic surfactant-based (niosomes) dehydration-rehydration vesicles (DRV). Using this procedure, it was shown that both these types of antigens can be effectively entrapped in DRV liposomes and DRV niosomes. The vesicle size of DRV niosomes was shown to be twice the diameter (~2µm) of that of their liposome counterparts. Incorporation of cryoprotectants such as sucrose in the DRV procedure resulted in reduced vesicle sizes while retaining high DNA incorporation efficiency (~95%). Transfection studies in COS 7 cells demonstrated that the choice of cationic lipid, the helper lipid, and the method of preparation, all influenced transfection efficiency indicating a strong interdependency of these factors. This phenomenon has been further reinforced when 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): cholesteryl 3b- [N-(N’ ,N’ -dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol)/DNA complexes were supplemented with non-ionic surfactants. Morphological analysis of these complexes using transmission electron microscopy and environmental scanning electron microscopy (ESEM) revealed the presence of heterogeneous structures which may be essential for an efficient transfection in addition to the fusogenic properties of DOPE. In vivo evaluation of these DNA incorporated vesicle systems in BALB/c mice showed weak antibody and cell-mediated immune (CMI) responses. Subsequent mock challenge with hepatitis B antigen demonstrated that, 1-monopalmitoyl glycerol (MP) based DRV, is a more promising DNA vaccine adjuvant. Studying these DRV systems as adjuvants for the Hepatitis B subunit antigen (HBsAg) revealed a balanced antibody/CMI response profile on the basis of the HBsAg specific antibody and cytokine responses which were higher than unadjuvated antigen. The effect of addition of MP, cholesterol and trehalose 6,6’-dibehenate (TDB) on the stability and immuno-efficacy of dimethyldioctadecylammonium bromide (DDA) vesicles was investigated. Differential scanning calorimetry showed a reduction in transition temperature of DDA vesicles by ~12°C when incorporated with surfactants. ESEM of MP based DRV system indicated an increased vesicle stability upon incorporation of antigen. Adjuvant activity of these systems tested in C57BL/6j mice against three subunit antigens i.e., mycobacterial fusion protein- Ag85B-ESAT-6, and two malarial antigens - merozoite surface protein-1, (MSP1), and glutamate rich protein, (GLURP) revealed that while MP and DDA based systems induced comparable antibody responses, DDA based systems induced powerful CMI responses.

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.

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.

Structure-activity relationships for α-calcitonin gene-related peptide

Calcitonin gene-related peptide (CGRP) is a member of the calcitonin (CT) family of peptides. It is a widely distributed neuropeptide implicated in conditions such as neurogenic inflammation. With other members of the CT family, it shares an N-terminal disulphide-bonded ring which is essential for biological activity, an area of potential α-helix, and a C-terminal amide. CGRP binds to the calcitonin receptor-like receptor (CLR) in complex with receptor activity-modifying protein 1 (RAMP1), a member of the family B (or secretin-like) GPCRs. It can also activate other CLR or calcitonin-receptor/RAMP complexes. This 37 amino acid peptide comprises the N-terminal ring that is required for receptor activation (residues 1-7); an α-helix (residues 8-18), a region incorporating a β-bend (residues 19-26) and the C-terminal portion (residues 27-37), that is characterized by bends between residues 28-30 and 33-34. A few residues have been identified that seem to make major contributions to receptor binding and activation, with a larger number contributing either to minor interactions (which collectively may be significant), or to maintaining the conformation of the bound peptide. It is not clear if CGRP follows the pattern of other family B GPCRs in binding largely as an α-helix. Linked Articles This article is part of a themed section on Neuropeptides. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.170.issue-7 © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23-60 of the calcitonin receptor-like receptor

The calcitonin receptor-like receptor (CLR) acts as a receptor for the calcitonin gene-related peptide (CGRP) but in order to recognize CGRP, it must form a complex with an accessory protein, receptor activity modifying protein 1 (RAMP1). Identifying the protein/protein and protein/ligand interfaces in this unusual complex would aid drug design. The role of the extreme N-terminus of CLR (Glu23-Ala60) was examined by an alanine scan and the results were interpreted with the help of a molecular model. The potency of CGRP at stimulating cAMP production was reduced at Leu41Ala, Gln45Ala, Cys48Ala and Tyr49Ala; furthermore, CGRP-induced receptor internalization at all of these receptors was also impaired. Ile32Ala, Gly35Ala and Thr37Ala all increased CGRP potency. CGRP specific binding was abolished at Leu41Ala, Ala44Leu, Cys48Ala and Tyr49Ala. There was significant impairment of cell surface expression of Gln45Ala, Cys48Ala and Tyr49Ala. Cys48 takes part in a highly conserved disulfide bond and is probably needed for correct folding of CLR. The model suggests that Gln45 and Tyr49 mediate their effects by interacting with RAMP1 whereas Leu41 and Ala44 are likely to be involved in binding CGRP. Ile32, Gly35 and Thr37 form a separate cluster of residues which modulate CGRP binding. The results from this study may be applicable to other family B GPCRs which can associate with RAMPs.

The role of ECL2 in CGRP receptor activation:a combined modelling and experimental approach

The calcitonin gene-related peptide (CGRP) receptor is a complex of a cal-citonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271-294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed. © 2013 The Authors.

Investigation of the activation mechanism and structural characterization of the CGRP receptor

The calcitonin gene-related peptide (CGRP) receptor is an unusual G protein-coupled receptor (GPCR) in that it comprises the calcitonin receptor-like receptor (CLR), receptor activity modifying protein 1 (RAMP1) and the receptor component protein (RCP). The RAMP1 has two other homologues – RAMP2 and RAMP3. The endogenous ligand for this receptor is CGRP, a 37 amino acid neuropeptide that act as a vasodilator. This peptide has been implicated in the aetiology of health conditions such as inflammation, Reynaud’s disease and migraine. A clear understanding of the mode of activation of this receptor could be key in developing therapeutic agents for associated health conditions. Although the crystal structure of the N-terminal extracellular domain (ECD) of this receptor (in complex with an antagonist) has been published, the details of receptor-agonist interactions at this domain, and so ultimately the mechanism of receptor activation, are still unclear. Also, the C-terminus of the CLR (in the CGRP receptor), especially around the presumed helix 8 (H8) region, has not been well studied for its role in receptor signalling. This research project investigated these questions. In this study, certain residues making up the putative N-terminal ligand-binding core of the CLR (in the CGRP receptor) were mapped out and found to be crucial for receptor signalling. They included W69 and D70 of the WDG motif in family B GPCRs, as well as Y91, F92, D94 and F95 in loop 2 of CLR N-terminus. Also, F163 at the cytoplasmic end of TM1 and certain residues spanning H8 and associated C-terminal region of CLR were found to be required for CGRP receptor signalling. These residues were investigated by site-directed mutagenesis where they were mutated to alanine (or other residues in specific cases) and the effect of the mutations on receptor pharmacology assessed by evaluating cAMP production, cell surface expression, total cell expression and aCGRP-mediated receptor internalization. Moreover, the N-terminal ECDs of the CLR and RAMPs (RAMP1, RAMP2 and RAMP3) were produced in a yeast host strain (Pichia pastoris) for the purpose of structural interaction study by surface plasmon resonance (SPR). Following expression and purification, these receptor proteins were found to individually retain their secondary structures when analysed by circular dichroism (CD). Results were analysed and interpreted with the knowledge of the secretin family receptor paradigm. The research described in this thesis has produced novel data that contributes to a clearer understanding of CGRP receptor pharmacology. The study on CLR and RAMPs ECDs could be a useful tool in determining novel interacting GPCR partners of RAMPs.