5-Hydroxytryptamine induced excitation and inhibition in the subthalamic nucleus:action at 5-HT2C, 5-HT4 and 5-HT1A receptors

Extracellular single-unit recordings in mouse brain slices were used to determine the effect of exogenously applied 5-HT on STN neurones. Recordings were made from 74 STN cells which fired action potentials at a regular rate of 7.19 ± 0.5 Hz. In 61 cells (82%), 5-HT application increased STN neurone firing rate (10 μM, 180 ± 16.8%, n = 35) with an estimated EC 50 of 5.4 μM. The non-specific 5-HT2 receptor agonist α-methyl 5-HT (1-10 μM) mimicked 5-HT induced excitations (15 cells). These excitations were significantly reduced by pre-perfusion with the specific 5-HT2C receptor antagonist RS102221 (500 nM, 9 cells) and the 5HT4 antagonist GR113808 (500 nM, 7 cells). In 6 cells (8%) 5-HT induced biphasic responses where excitation was followed by inhibition, while in 7 cells (9%) inhibition of firing rate was observed alone. Inhibitory responses were reduced by the 5-HT1A antagonist WAY100135 (1 μM, 4 cells). No inhibitory responses were observed following α-methyl 5-HT applications. Both the excitations and inhibitions were unaffected by picrotoxin (50 μM, n = 5) and CNQX (10 μM, n = 5) indicative of direct postsynaptic effects. Thus, in STN neurones, 5-HT elicits two distinct effects, at times on the same neurone, the first being an excitation which is mediated by 5-HT 2C and 5-HT4 receptors and the second an inhibition which is mediated by 5-HT1A receptors. © 2005 Elsevier Ltd. All rights reserved.

Meningiomas expressing and responding to cholecystokinin (CCK)

The effect of cholecystokinin (CCK) on cultured human meningioma derived cells was investigated. Exposure of meningioma cells for 6-12 days to CCK-8s (2-200 nM) resulted in a dose dependent stimulation of cell growth to a maximum of 1.1-fold over basal controls. A time course study showed stimulation of cell growth at day 3 followed by increase throughout day 6. The stimulatory effect of CCK on meningioma cell growth was completely abolished by a CCK-B specific receptor antagonist, L-365,260. Reverse-transcription of meningioma-derived RNA into cDNA followed by amplification by the polymerase chain reaction using specific primers for CCK peptide and its CCK-A and/B receptor revealed 100% presence of CCK peptide and CCK-B receptors mRNA whereas CCK-A receptor was expressed in 66% of the meningiomas. These results provide evidence that human meningioma cells possess CCK peptide and its receptors the activation of which leads to increase of cell growth possibly via an autocrine/paracrine mechanism. © Springer 2005.

The role of Ca2+ in the generation of spontaneous astrocytic Ca2+ oscillations

Astrocytes in the rat thalamus display spontaneous [Ca2+]i oscillations that are due to intracellular release, but are not dependent on neuronal activity. In this study we have investigated the mechanisms involved in these spontaneous [Ca2+]i oscillations using slices loaded with Fluo-4 AM (5 μM) and confocal microscopy. Bafilomycin A1 incubation had no effect on the number of spontaneous [Ca2+]i oscillations indicating that they were not dependent on vesicular neurotransmitter release. Oscillations were also unaffected by ryanodine. Phospholipase C (PLC) inhibition decreased the number of astrocytes responding to metabotropic glutamate receptor (mGluR) activation but did not reduce the number of spontaneously active astrocytes, indicating that [Ca2+]i increases are not due to membrane-coupled PLC activation. Spontaneous [Ca2+]i increases were abolished by an IP3 receptor antagonist, whilst the protein kinase C (PKC) inhibitor chelerythrine chloride prolonged their duration, indicating a role for PKC and inositol 1,4,5,-triphosphate receptor activation. BayK8644 increased the number of astrocytes exhibiting [Ca2+]i oscillations, and prolonged the responses to mGluR activation, indicating a possible effect on store-operated Ca2+ entry. Increasing [Ca2+]o increased the number of spontaneously active astrocytes and the number of transients exhibited by each astrocyte. Inhibition of the endoplasmic reticulum Ca2+ ATPase by cyclopiazonic acid also induced [Ca2+]i transients in astrocytes indicating a role for cytoplasmic Ca2+ in the induction of spontaneous oscillations. Incubation with 20 μM Fluo-4 reduced the number of astrocytes exhibiting spontaneous increases. This study indicates that Ca2+ has a role in triggering Ca2+ release from an inositol 1,4,5,-triphosphate sensitive store in astrocytes during the generation of spontaneous [Ca2+]i oscillations

New drugs for type 2 diabetes mellitus:what is their place in therapy?

Oral therapy for type 2 diabetes mellitus, when used appropriately, can safely assist patients to achieve glycaemic targets in the short to medium term. However, the progressive nature of type 2 diabetes usually requires a combination of two or more oral agents in the longer term, often as a prelude to insulin therapy. Issues of safety and tolerability, notably weight gain, often limit the optimal application of anti-diabetic drugs such as sulforylureas and thiazolidinediones. Moreover, the impact of different drugs, even within a single class, on the risk of long-term vascular complications has come under scrutiny. For example, recent publication of evidence suggesting potential detrimental effects of rosiglitazone on myocardial events generated a heated debate and led to a reduction in use of this drug. In contrast, current evidence supports the view that pioglitazone has vasculoprotective properties. Both drugs are contraindicated in patients who are at risk of heart failure. An additional recently identified safety concern is an increased risk of fractures, especially in postmenopausal women. Several new drugs with glucose-lowering efficacy that may offer certain advantages have recently become available. These include (i) injectable glucagonlike peptide-1 (GLP-1) receptor agonists and oral dipeptidyl peptidase-4 (DPP-4) inhibitors; (ii) the amylin analogue pramlintide; and (iii) selective cannabinoid receptor-1 (CB1) antagonists. GLP-1 receptor agonists, such as exenatide, stimulate nutrient-induced insulin secretion and reduce inappropriate glucagon secretion while delaying gastric emptying and reducing appetite. These agents offer a low risk of hypoglycaemia combined with sustained weight loss. The DPP-4 inhibitors sitagliptin and vildagliptin are generally weight neutral, with less marked gastrointestinal adverse effects than the GLP-1 receptor agonists. Potential benefits of GLP-1 receptor stimulation on P cell neogenesis are under investigation. Pancreatitis has been reported in exenatide-treated patients. Pramlintide, an injected peptide used in combination with insulin, can reduce insulin dose and bodyweight. The CB1 receptor antagonist rimonabant promotes weight loss and has favourable effects on aspects of the metabolic syndrome, including the hyperglycaemia of type 2 diabetes. However, in 2007 the US FDA declined approval of rimonabant, requiring more data on adverse effects, notably depression. The future of dual peroxisome proliferator-activated receptor-alpha/gamma agonists, or glitazars, is presently uncertain following concerns about their safety. In conclusion, several new classes of drugs have recently become available in some countries that offer new options for treating type 2 diabetes. Beneficial or neutral effects on bodyweight are an attractive feature of the new drugs. However, the higher cost of these agents, coupled with an absence of long-term safety and clinical outcome data, need to be taken into consideration by clinicians and healthcare organizations.

Antagonistic effects of two novel GIP analogs, (Hyp3)GIP and (Hyp3)GIPLys16PAL, on the biological actions of GIP and longer-term effects in diabetic ob/ob mice

This study examines the actions of the novel enzyme-resistant, NH 2-terminally modified GIP analog (Hyp3)GIP and its fatty acid-derivatized analog (Hyp3)GIPLys16PAL. Acute effects are compared with the established GIP receptor antagonist (Pro3)GIP. All three peptides exhibited DPP IV resistance, and significantly inhibited GIP stimulated cAMP formation and insulin secretion in GIP receptor-transfected fibroblasts and in clonal pancreatic BRIN-BD11 cells, respectively. Likewise, in obese diabetic ob/ob mice, intraperitoneal administration of GIP analogs significantly inhibited the acute antihyperglycemic and insulin-releasing effects of native GIP. Administration of once daily injections of (Hyp 3)GIP or (Hyp3)GIPLys16PAL for 14 days resulted in significantly lower plasma glucose levels (P < 0.05) after (Hyp 3)GIP on days 12 and 14 and enhanced glucose tolerance (P < 0.05) and insulin sensitivity (P < 0.05 to P < 0.001) in both groups by day 14. Both (Hyp3)GIP and (Hyp3)GIPLys16PAL treatment also reduced pancreatic insulin (P < 0.05 to P < 0.01) without affecting islet number. These data indicate that (Hyp3)GIP and (Hyp 3)GIPLys16PAL function as GIP receptor antagonists with potential for ameliorating obesity-related diabetes. Acylation of (Hyp 3)GIP to extend bioactivity does not appear to be of any additional benefit. Copyright © 2007 the American Physiological Society.

Pharmacological discrimination of calcitonin receptor:receptor activity-modifying protein complexes

Calcitonin (CT) receptors dimerize with receptor activity-modifying proteins (RAMPs) to create high-affinity amylin (AMY) receptors, but there is no reliable means of pharmacologically distinguishing these receptors. We used agonists and antagonists to define their pharmacology, expressing the CT (a) receptor alone or with RAMPs in COS-7 cells and measuring cAMP accumulation. Intermedin short, otherwise known as adrenomedullin 2, mirrored the action of αCGRP, being a weak agonist at CT(a), AMY 2(a), and AMY3(a) receptors but considerably more potent at AMY1(a) receptors. Likewise, the linear calcitonin gene-related peptide (CGRP) analogs (Cys(ACM)2,7)hαCGRP and (Cys(Et) 2,7)haCGRP were only effective at AMY1(a) receptors, but they were partial agonists. As previously observed in COS-7 cells, there was little induction of the AMY2(a) receptor phenotype; thus, AMY 2(a) was not examined further in this study. The antagonist peptide salmon calcitonin8-32 (sCT8-32) did not discriminate strongly between CT and AMY receptors; however, AC187 was a more effective antagonist of AMY responses at AMY receptors, and AC413 additionally showed modest selectivity for AMY1(a) over AMY3(a) receptors. CGRP8-37 also demonstrated receptor-dependent effects. CGRP 8-37 more effectively antagonized AMY at AMY1(a) than AMY3(a) receptors, although it was only a weak antagonist of both, but it did not inhibit responses at the CT(a) receptor. Low CGRP 8-37 affinity and agonism by linear CGRP analogs at AMY 1(a) are the classic signature of a CGRP2 receptor. Our data indicate that careful use of combinations of agonists and antagonists may allow pharmacological discrimination of CT(a), AMY1(a), and AMY3(a) receptors, providing a means to delineate the physiological significance of these receptors. Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics.

A key role for transmembrane prolines in calcitonin receptor-like receptor agonist binding and signalling:implications for family B G-protein-coupled receptors

Calcitonin receptor like-receptor is a family B G-protein coupled receptor (GPCR). It requires receptor activity modifying protein (RAMP) 1 to give a calcitonin gene-related peptide (CGRP) receptor. Little is known of how members of this receptor family function. Proline residues often form important kinks in alpha-helices. Therefore, all proline residues within the transmembrane helices of the receptor (Pro241, Pro244 in helix 4, Pro275 in helix 5, Pro321 and Pro331 in helix 6) were mutated to alanine. Pro241 Pro275, and Pro321 are highly conserved throughout all family B GPCRs. The binding of CGRP and its ability to stimulate cAMP production were investigated in mutant and wild-type receptors after transient transfection into COS-7 cells with RAMP1. The P321A mutation significantly decreased the pEC(50) for CGRP and reduced its affinity but did not change cell-surface expression. Antagonist binding [CGRP(8-37) and 1-piperidinecarboxamide N-[2-[[5amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3 5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quina zolinyl) (BIBN4096BS)] was little altered by the mutation. Adrenomedullin-mediated signaling was disrupted when P321A was coexpressed with RAMP1, RAMP2, or RAMP3. The P331A mutant produced a moderate reduction in CGRP binding and receptor activation. Mutation of the other residues had no effect on receptor function. Thus, Pro321 and Pro331 are required for agonist binding and receptor activation. Modeling suggested that Pro321 induces a bend in helix 6, bringing its C terminus near that of helix 3, as seen in many family A GPCRs. This is abolished in P321A. P321A-I325P predicted to restore this conformation, showed wild-type activation. Modeling can also rationalize the effects of transmembrane proline mutants previously reported for another family B GPCR, the VPAC(1) receptor.

The selectivity and structural determinants of peptide antagonists at the CGRP receptor of rat, L6 myocytes

1. Potency orders were determined for a series of agonists and antagonists on the calcitonin gene-related peptide (CGRP) receptor of rat L6 myocytes. The agents tested were all shown to have been active against CGRP, amylin or adrenomedullin receptors. 2. AC187 had a PIC50 Of 6.8 ± 0.10, making it 14 fold less potent as an antagonist than CGRP8-37 (PIC50, 7.95 ± 0.14). Amyline8-37 was equipotent to AC187 (PIC50, 6.6 ± 0.16) and CGRP19-37 was a fold less potent than either (pIC50 6.1 ± 0.24). 3. [Ala11]-CGRP8-37 was 6 fold less potent than CGRP8-37, (pIC50 7.13 ± 0.14), whereas [Ala18] CGRP8-37 was approximately equipotent to CGRP8-37 (pIC50, 7.52 ± 0.15). However, [Ala11,Ala18]- CGRP8-37 was over 300 fold less potent than CGRP8-37 (pIC50, 5.30 ± 0.04). 4. [Tyr0]-CGRP28-37, amylin19-37 and adrenomedullin22-52 were inactive as antagonists at concentrations of up to 1 μM. 5. Biotinyl-human α-CGRP was 150 fold less potent than human α-CGRP itself (EC50 values of 48 ± 17 nM and 0.31 ± 0.13 nM, respectively). At 1 μM, [Cys(acetomethoxy)(2'7)]-CGRP was inactive as an agonist. 6. These results confirm a role for Arg11 in maintaining the high affinity binding of CGRP8-37. Arg18 is of less direct significance for high affinity binding, but it may be important in maintaining the amphipathic nature of CGRP and its analogues.

Pharmacological characterization of a receptor for calcitonin gene-related peptide on rat, L6 myocytes

1 The L6 myocyte cell line expresses high affinity receptors for calcitonin gene-related peptide (CGRP) which are coupled to activation of adenylyl cyclase. The biochemical pharmacology of these receptors has been examined by radioligand binding or adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation. 2 In intact cells at 37 degrees C, human and rat alpha- and beta-CGRP all activated adenylyl cyclase with EC50s of about 1.5 nM. A number of CGRP analogues containing up to five amino acid substitutions showed similar potencies. In membrane binding studies at 22 degrees C in 1 mM Mg2+, the above all bound to a single site with IC50s of 0.1-0.4 nM. 3 The fragment CGRP(8-37) acted as a competitive antagonist of CGRP stimulation of adenylyl cyclase with a calculated Kd of 5 nM. The Kd determined in membrane binding assays was lower (0.5 nM). 4 The N-terminal extended human alpha-CGRP analogue Tyro-CGRP activated adenylyl cyclase and inhibited [125I]-iodohistidyl-CGRP binding less potently than human alpha-CGRP (EC50 for cyclase = 12 nM, IC50 for binding = 4 nM). 5 The pharmacological profile of the L6 CGRP receptor suggests that it most closely resembles sites on skeletal muscle, cardiac myocytes and hepatocytes. The L6 cell line should be a stable homogeneous model system in which to study CGRP mechanisms and pharmacology."

C-reactive protein mediates CD11b expression in monocytes through the non-receptor tyrosine kinase, Syk, and calcium mobilization but not through cytosolic peroxides

Objective: C-Reactive protein (CRP) can modulate integrin surface expression on monocytes following Fcγ receptor engagement. We have investigated the signal transduction events causing this phenotypic alteration. Methods: CRP-induced signalling events were examined in THP-1 and primary monocytes, measuring Syk phosphorylation by Western blotting, intracellular Ca2+ ([Ca2+]i) by Indo-1 fluorescence and surface expression of CD11b by flow cytometry. Cytosolic peroxides were determined by DCF fluorescence. Results: CRP induced phosphorylation of Syk and an increase in [Ca2+]i both of which were inhibitable by the Syk specific antagonist, piceatannol. Piceatannol also inhibited the CRP-induced increase in surface CD11b. In addition, pre-treatment of primary monoytes with the Ca2+ mobiliser, thapsigargin, increased CD11b expression; this effect was accentuated in the presence of CRP but was abolished in the presence of the [Ca2+]i chelator, BAPTA. CRP also increased cytosolic peroxide levels; this effect was attenuated by antioxidants (ascorbate, α-tocopherol), expression of surface CD11b not being inhibited by antioxidants alone. Conclusion: CRP induces CD11b expression in monocytes through a peroxide independent pathway involving both Syk phosphorylation and [Ca2+]i release. © Birkhäuser Verlag, 2005.

Toward the discovery of vaccine adjuvants:coupling in silico screening and in vitro analysis of antagonist binding to human and mouse CCR4 receptors

Background Adjuvants enhance or modify an immune response that is made to an antigen. An antagonist of the chemokine CCR4 receptor can display adjuvant-like properties by diminishing the ability of CD4+CD25+ regulatory T cells (Tregs) to down-regulate immune responses. Methodology Here, we have used protein modelling to create a plausible chemokine receptor model with the aim of using virtual screening to identify potential small molecule chemokine antagonists. A combination of homology modelling and molecular docking was used to create a model of the CCR4 receptor in order to investigate potential lead compounds that display antagonistic properties. Three-dimensional structure-based virtual screening of the CCR4 receptor identified 116 small molecules that were calculated to have a high affinity for the receptor; these were tested experimentally for CCR4 antagonism. Fifteen of these small molecules were shown to inhibit specifically CCR4-mediated cell migration, including that of CCR4+ Tregs. Significance Our CCR4 antagonists act as adjuvants augmenting human T cell proliferation in an in vitro immune response model and compound SP50 increases T cell and antibody responses in vivo when combined with vaccine antigens of Mycobacterium tuberculosis and Plasmodium yoelii in mice.

Depolarisation and suppression of burst firing activity in the mouse subthalamic nucleus by dopamine D1/D5 receptor activation of a cyclic-nucleotide gated non-specific cation conductance

Neuronal burst firing in the subthalamic nucleus (STN) is one of the hallmarks of dopamine depletion in Parkinson's disease. Here, we have determined the postsynaptic effects of dopamine in the STN and the functional consequences of dopamine receptor modulation on burst firing in vitro. STN cells displayed regular spiking activity at a rate of 7.9 +/- 0.5 Hz. Application of dopamine (30 mu M) induced membrane depolarisations accompanied by an increase in firing rate of mean 12.0 +/- 0.6 Hz in all 69 cells. The dopamine effect was mimicked by the dopamine D1/D5 receptor agonist SKF38393 (10 mu M, 17 cells) and the dopamine D2-like receptor agonist quinpirole (10 mu M, 35 cells), partly reduced by D1/D5 antagonist SCH23390 (2 mu M, seven cells), but unaffected by the D2 antagonists sulpiride (10 mu M, seven cells) or eticlopride (10 mu M, six cells). Using voltage ramps, dopamine induced an inward current of 69 +/- 9.4 pA at a holding potential of -60 mV (n = 17). This current was accompanied by an increase in input conductance of 1.55 +/- 0.35 nS which reversed at -30.6 +/- 2.3 mV, an effect mimicked by SKF38393 (10 AM, nine cells). Similar responses were observed when measuring instantaneous current evoked by voltage steps and in the presence of the I-h blocker, ZD7288, indicating effects independent of I-h. The increase in conductance was blocked by SCH23390 (2 mu M, n = 4), mimicked by the activator of adenylyl cyclase forskolin (10 mu M, n = 7) and blocked by H-89, an inhibitor of cyclic AMP dependent protein kinase A (10 PM, n = 6). These results indicate that the dopamine depolarisation is in part mediated by D1/D5 receptor mediated activation of a cyclic-nucleotide gated (CNG) non-specific cation conductance. This conductance contributes to the membrane depolarisation that changes STN neuronal bursting to more regular activity by significantly increasing burst duration and number of spikes per burst.

The heteromeric 5-HT3A/B receptor:the effect of 5-HT3B subunits on receptor structure and ligand recognition

The 5-HT3 receptors are members of the cys-loop family of ligand-gated ion channels. Two functional subtypes are known, the homomeric 5HT3A and the heteromeric 5HT3A/B receptors, which exhibit distinct biophysical characteristics but are difficult to differentiate pharmacologically. Atomic force microscopy has been used to determine the stoichiometry and architecture of the heteromeric 5HT3A/B receptor. Each subunit was engineered to express a unique C-terminal epitope tag, together with six sequential histidine residues to facilitate nickel affinity purification. The 5-HT3 receptors, ectopically expressed in HEK293 cells, were solubilised, purified and decorated with antibodies to the subunit specific epitope tags. Imaging of individual receptors by atomic force microscopy revealed a pentameric arrangement of subunits in the order BBABA, reading anti-clockwise when viewed from the extracellular face. Homology models for the heteromeric receptor were then constructed using both the electron microscopic structure of the nicotinic acetylcholine receptor, from Torpedo marmorata, and the X-ray crystallographic structure of the soluble acetylcholine binding protein, from Lymnaea stagnalis, as templates. These homology models were used, together with equivalent models constructed for the homomeric receptor, to interpret mutagenesis experiments designed to explore the minimal recognition differences of both the natural agonist, 5-HT, and the competitive antagonist, granisetron, for the two human receptor subtypes. The results of this work revealed that the 5-HT3B subunit residues within the ligand binding site, for both the agonist and antagonist, are accommodating to conservative mutations. They are consistent with the view that the 5-HT3A subunit provides the principal and the 5-HT38 subunit the complementary recognition interactions at the binding interface.

Non-peptidic antagonists of the CGRP receptor, BIBN4096BS and MK-0974, interact with the calcitonin receptor-like receptor via methionine-42 and RAMP1 via tryptophan-74

The receptor for calcitonin gene-related peptide (CGRP) has been the target for the development of novel small molecule antagonists for the treatment of migraine. Two such antagonists, BIBN4096BS and MK-0974, have shown great promise in clinical trials and hence a deeper understanding of the mechanism of their interaction with the receptor is now required. The structure of the CGRP receptor is unusual since it is comprised of a hetero-oligomeric complex between the calcitonin receptor-like receptor (CRL) and an accessory protein (RAMP1). Both the CLR and RAMP1 components have extracellular domains which interact with each other and together form part of the peptide-binding site. It seems likely that the antagonist binding site will also be located on the extracellular domains and indeed Trp-74 of RAMP1 has been shown to form part of the binding site for BIBN4096BS. However, despite a chimeric study demonstrating the role of the N-terminal domain of CLR in antagonist binding, no specific residues have been identified. Here we carry out a mutagenic screen of the extreme N-terminal domain of CLR (residues 23-63) and identify a mutant, Met-42-Ala, which displays 48-fold lower affinity for BIBN4096BS and almost 900-fold lower affinity for MK-0974. In addition, we confirm that the Trp-74-Lys mutation at human RAMP1 reduces BIBN4096BS affinity by over 300-fold and show for the first time a similar effect for MK-0974 affinity. The data suggest that the non-peptide antagonists occupy a binding site close to the interface of the N-terminal domains of CLR and RAMP1.

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.