CGRP 27-37 analogues with high affinity to the CGRP1 receptor show antagonistic properties in a rat blood flow assay

CGRP Y0-28-37 is known as a selective CGRP1 receptor antagonist. We succeeded in optimising the CGRP1 receptor affinity of this fragment by multiple amino acid replacement. The analogues [p34, F35]CGRP 27-37 and [D31, p34, F35]CGRP 27-37 exhibit a 100-fold increased affinity compared to the unmodified segment. Receptor binding studies were performed with human neuroblastoma cells SK-N-MC, which selectively express the hCGRP1 receptor. Blood flow, which is increased by exogenous CGRP, was measured in the right femoral artery. Preincubation of the rats with [p34, F35]CGRP 27-37 and [D31, p34, F35]CGRP 27-37 led to a significant decrease in CGRP induced increase in vascular conductance indicating the antagonistic properties of these compounds. Interestingly, an exchange of the amino acid Asn31 to Asp31 in [p34, F35]CGRP 27-37 shortened the period of the antagonistic effect significantly, suggestive of a different rate of metabolism for the two ligands. Secondary structure investigations obtained by circular dichroism measurements revealed that an increase in ordered structure correlates with high binding affinity.     

C-terminal peptides of calcitonin gene-related peptide act as agonists at the cholecystokinin receptor

We have previously described that [Tyr⁰]CGRP(28–37) acts as a receptor antagonist of rat CGRP in guinea pig pancreatic acini. We therefore examined other C-terminal peptides of CGRP for such activity. CGRP-acetyl(28–37) acetate did act as a rat CGRP antagonist. However, C-terminal CGRP peptides of 4 to 8 amino acid residues did not antagonize the actions of rat CGRP but stimulated amylase secretion. In pancreatic acini, a maximally effective concentration of rat CGRP (100 nM) caused a 2.1-fold increase in amylase secretion. When the C-terminal peptides of CGRP were tested in at 100 μM, CGRP(34–37) caused a 1.8-fold increase in amylase secretion, CGRP(33–37) a 2.8-fold increase, CGRP(32–37) a 9.2-fold increase, CGRP(31–37) a 4.1-fold increase, and CGRP(30–37) a 5.1-fold increase. Further studies with the most effective peptide, CGRP(32–37), demonstrated that it did not cause release of lactate dehydrogenase, and thus did not cause amylase release by cell damage. Unlike rat CGRP, CGRP(32–37) did not increase cellular cyclic AMP, but did stimulate outflux of ⁴⁵Ca. CGRP(32–37)-stimulated amylase release was not inhibited by the substance P receptor antagonist, spantide, by the bombesin receptor antagonist, [D-Phe⁶]bombesin(6–13) propylamide, or by the muscarinic receptor antagonist, atropine, but was inhibited by the CCK receptor antagonist L364,718. C-terminal peptides of CGRP inhibited binding of ¹²⁵I-BH-CCK-8, with the relative potencies of the peptides being the same as their relative potencies for stimulating amylase secretion. The present data demonstrate that C-terminal peptides of CGRP, although they have only 2 amino acid residues in common with CCK(26–33), act exclusively at CCK receptors on pancreatic acini to stimulate amylase secretion.     

Investigation of the species selectivity of a nonpeptide CGRP receptor antagonist using a novel pharmacodynamic assay

The recent discovery of several nonpeptide CGRP antagonists have led to significant advances in our understanding of CGRP receptor pharmacology. Specifically, these antagonists have demonstrated a clear species selectivity with >100-fold greater affinity for human CGRP receptor compared to receptors from other species, such as rat, rabbit and guinea pig. Therefore, nonhuman primate models are required to accurately assess the in vivo activity of these antagonists. The commonly used model in marmosets involves electrical stimulation of the trigeminal ganglia and is a technically difficult and terminal procedure. In this report, we describe a noninvasive pharmacodynamic model in which topical application of capsaicin is utilized to induce the release of endogenous CGRP and a vasodilatory response which can be measured using laser Doppler imaging. Using the potent and selective CGRP antagonist Compound 3, which is an analog of the well-characterized compound BIBN4096BS, we demonstrated 62% inhibition with 300 microg/kg, i.v., in the rat. When tested in the rhesus monkey, only 30 microg/kg of Compound 3 was needed to produce complete inhibition, suggesting that the rhesus CGRP receptor shares a pharmacological profile similar to marmoset and human receptors. Two separate measurements were obtained in this model to provide an indication of both the acute inhibitory effect as well as the prophylactic effect of the CGRP antagonist. At the doses studied, Compound 3 was equally effective on both the acute and prophylactic inhibition of CGRP-mediated vasodilation in rat and rhesus. In conclusion, this is the first report to describe and validate a noninvasive model in nonhuman primates that allows rapid evaluation of CGRP antagonist activity against endogenous CGRP.     

Proceedings of the 4th International Meeting on Calcitonin Gene-Related Peptide and its Receptor

Calcitonin Gene-Related Peptide (CGRP), a 37 amino acid peptide identified as the alternately spliced gene product of calcitonin gene, is a sensory neuropeptide with potent cardiovascular effects. CGRP is distributed throughout the central and peripheral nervous systems and possesses diverse biological actions. CGRP has been suggested to play a role in diseases such as migraine, diabetes, pain, and inflammation. Two forms of CGRP (alpha and beta) that differ in three amino acids have been identified and are encoded by different genes. Based on the differential biological activities of various CGRP analogs, the CGRP receptors have been classified into CGRP1 and CGRP2. Structure-activity studies of CGRP analogs showed that the C- and N-terminal regions of the peptide interact independently with their receptors. While C-terminal peptide, CGRP (8-37) behaves as a CGRP1 receptor antagonist, N-terminal peptide CGRP (1-12) behaves as a weak agonist. Structural modifications of CGRP(28-37) have yielded micromolar to nanomolar affinity ligands. CGRP receptor belongs to the calcitonin receptor like receptor (CRLR) family of G-protein-coupled receptors and has been shown to require a single transmembrane domain protein called receptor activity modifying protein-1 (RAMP1) for its functional expression as well as activity. Human, rat, and porcine CRLRs have been cloned and characterized. Currently, the major focus is on the identification of potent and specific nonpeptide antagonists for this receptor in order to understand the physiological and pathophysiological role of this peptide.     

Identification and pharmacological characterization of domains involved in binding of CGRP receptor antagonists to the calcitonin-like receptor

The calcitonin-like receptor (CLR) and the calcitonin receptor (CTR) interact with receptor activity-modifying protein 1 (RAMP1) at the cell surface to form heterodimeric receptor complexes. CLR and CTR are members of the class II (family B) G-protein-coupled receptors (GPCR) and bind calcitonin gene-related peptide (CGRP) with similar affinities when coexpressed with RAMP1. The observation that various nonpeptide CGRP receptor antagonists display a higher affinity for the CLR/RAMP1 complex than for CTR/RAMP1 provided an opportunity to investigate the molecular determinants of the differential receptor affinities of these antagonists. A chimeric receptor approach was utilized to identify key domains within CLR responsible for conferring high-affinity antagonist binding. Initial chimera experiments implicated distinct regions within CLR as responsible for the affinities of structurally diverse CGRP receptor antagonists. Dissection of these key regions implicated amino acids 37-63 located in the amino terminus of CLR as responsible for the high-affinity interaction of one structural class, while transmembrane domain (TM) 7 was responsible for the interaction of a second class of antagonist. A unique binding interaction in the amino terminus of CLR is consistent with the observation that these compounds also interact with the extracellular region of RAMP1 and could suggest the formation of a binding pocket between the two proteins. Conversely, a compound which interacted with TM7 did not display a similar RAMP1 dependence, suggesting an allosteric mechanism of antagonism. Collectively, these data provide insight into two alternative mechanisms of antagonism for this unique heterodimeric receptor complex.     

Discovery of (R)-N-(3-(7-methyl-1H-indazol-5-yl)-1-(4-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidine-1-carboxamide (BMS-742413): A potent human CGRP antagonist with superior safety profile for the treatment of migraine through intranasal delivery

Calcitonin gene-related peptide (CGRP) receptor antagonists have been shown to be efficacious as abortive migraine therapeutics with the absence of cardiovascular liabilities that are associated with triptans. Herein, we report the discovery of a highly potent CGRP receptor antagonist, BMS-742413, with the potential to provide rapid onset of action through intranasal delivery. The compound displays excellent aqueous solubility, oxidative stability, and toxicological profile. BMS-742413 has good intranasal bioavailability in the rabbit and shows a robust, dose-dependent inhibition of CGRP-induced increases in marmoset facial blood flow.     

Mechanisms of proton-induced stimulation of CGRP release from rat antrum

Mechanisms of acid-evoked CGRP release from gastric afferent nerves were investigated in rat antral mucosal/submucosal tissues. Low pH (pH 4.0, 5.0 and 6.0) stimulated antral CGRP release significantly and dose-dependently from rat antral fragments. Removal of extracellular calcium from the incubation medium resulted in significant inhibition (59%, P < 0.001) of acid (pH 4.0)-stimulated CGRP release. Conotoxin (1 x 10(-7) M), the selective blocker of N-type calcium channels, also significantly inhibited proton (pH 4.0)-induced CGRP release to values that were 74% below net stimulated levels. Neither nifedipine (1 x 10(-6) M), the L-type Ca(2+)-channel antagonist, nor indomethacin (1 x 10(-5) M), inhibitor of prostaglandin synthesis, altered acid-induced CGRP release. In contrast, ruthenium red (1 x 10(-5) M), capsaicin antagonist, almost completely prevented acid (pH 4.0)-stimulated CGRP release. Capsazepine (1 x 10(-4) M), a specific capsaicin receptor antagonist, also completely abolished acid-induced CGRP release. In conclusion, the results of these studies indicate that hydrogen ions are capable of evoking CGRP release from peripheral sensory neurons in rat antral mucosal/submucosal tissues. Proton-evoked CGRP release requires extracellular calcium and involves N-type calcium channels. Furthermore, acid appears to exert a capsaicin-like effect to evoke sensory neuropeptide release that is sensitive to capsazepine and ruthenium red. These data suggest that proton-induced antral CGRP release represents a direct action of hydrogen ions on mucosal/submucosal sensory dendritic nerve endings to effect local release of neuropeptide.     

Identification of a novel RAMP-independent CGRP receptor antagonist

Identification of an HIV integrase inhibitor with micromolar affinity for the CGRP receptor led to the discovery of a series of structurally novel CGRP receptor antagonists. Optimization of this series produced compound 16, a low-molecular weight CGRP receptor antagonist with good pharmacokinetic properties in both rat and dog. In contrast to other nonpeptide antagonists, the activity of 16 was affected by the presence of divalent cations and showed evidence of an alternative, RAMP-independent CGRP receptor binding site.     

Receptor activity-modifying protein 1 determines the species selectivity of non-peptide CGRP receptor antagonists

The heterodimeric CGRP receptor requires co-expression of calcitonin receptor-like receptor (CRLR) and an accessory protein called receptor activity-modifying protein (RAMP) 1 (McLatchie, L. M., Fraser, N. J., Main, M. J., Wise, A., Brown, J., Thompson, N., Solari, R., Lee, M. G., and Foord, S. M. (1998) Nature 393, 333-339). Several non-peptide CGRP receptor antagonists have been shown to exhibit marked species selectivity, with >100-fold higher affinities for the human CGRP receptor than for receptors from other species (Doods, H., Hallermayer, G., Wu, D., Entzeroth, M., Rudolf, K., Engel, W., and Eberlein, W. (2000) Br. J. Pharmacol. 129, 420-423; Edvinsson, L., Sams, A., Jansen-Olesen, I., Tajti, J., Kane, S. A., Rutledge, R. Z., Koblan, K. S., Hill, R. G., and Longmore, J. (2001) Eur. J. Pharmacol. 415, 39-44). This observation provided an opportunity to map the determinants of receptor affinity exhibited by BIBN4096BS and the truncated analogs, Compounds 1 and 2. All three compounds exhibited higher affinity for the human receptor, human CRLR/human RAMP1, than for the rat receptor, rat CRLR/rat RAMP1. We have now demonstrated that this species selectivity was directed exclusively by RAMP1. By generating recombinant human/rat CRLR/RAMP1 receptors, we demonstrated that co-expression of human CRLR with rat RAMP1 produced rat receptor pharmacology, and vice versa. Moreover, with rat/human RAMP1 chimeras and site-directed mutants, we have identified a single amino acid at position 74 of RAMP1 that modulates the affinity of small molecule antagonists for CRLR/RAMP1. Replacement of lysine 74 in rat RAMP1 with tryptophan (the homologous amino acid in the human receptor) resulted in a > or =100-fold increase in antagonist affinities, similar to the K(i) values for the human receptor. These observations suggest that important determinants of small molecule antagonist affinity for the CGRP receptor reside within the extracellular region of RAMP1 and provide evidence that this receptor accessory protein may participate in antagonist binding.     

Mapping the CGRP receptor ligand binding domain: Tryptophan-84 of RAMP1 is critical for agonist and antagonist binding

The calcitonin receptor-like receptor (CLR) associates with the accessory protein RAMP1 to form a receptor for the neuropeptide calcitonin gene-related peptide (CGRP). Multiple lines of evidence have implicated CGRP in the pathophysiology of migraine headache making the CGRP receptor an attractive target for development of small-molecule antagonists as a novel treatment for this debilitating condition. The CGRP receptor antagonists telcagepant and olcegepant (BIBN4096BS) have demonstrated clinical efficacy in the treatment of migraine and there is now a need to better understand how these molecules interact with the receptor. Previous work has shown the extracellular portion of RAMP1 to be important for binding of these antagonists, with tryptophan-74 being a key interaction site. The crystal structure of the extracellular portion of human RAMP1 placed tryptophan-74 in a hydrophobic patch hypothesized to interact with CGRP receptor ligands and also identified nearby residues that may be important for ligand binding. In this study we explored the role played by these residues of RAMP1 using an alanine replacement strategy. We confirmed a role for tryptophan-74 in antagonist binding and also identified arginine-67 as being important for binding of telcagepant but not compound 3, a close analog of BIBN4096BS. We also identified tryptophan-84 as being critical for both high-affinity binding of the non-peptide antagonists as well as the peptides CGRP and CGRP(8-37). These data for the first time pinpoint a specific RAMP1 residue important for both antagonist and agonist potency and are consistent with the N-terminal domain of RAMP1 forming the binding pocket interface with CLR.     

Adrenomedullin inhibits neurotransmission of calcitonin gene-related peptide (CGRP)-containing vasodilator nerves in rat mesenteric resistance arteries.

We have reported that the rat mesenteric resistance artery has innervation of calcitonin gene-related peptide (CGRP)-containing vasodilator nerves (CGRPergic nerves). We also demonstrated that adrenomedullin (AM) causes mesenteric vasodilation through activation of CGRP receptors. The present study was designed to examine the effect of AM on neurotransmission of CGRPergic nerves in rat mesenteric arteries. In preconstricted preparations without endothelium, periarterial nerve stimulation (PNS, 1 and 2 Hz) induced a frequency-dependent vasodilation. A bolus injection of CGRP (10 pmol) into the perfusate also caused a vasodilation. AM (0.1 to 10 nM) concentration-dependently caused 40% to 60% inhibition of the PNS-induced vasodilation, but AM did not attenuate vasodilation induced by exogenous CGRP injection. The inhibitory effect of AM (10 nM) on PNS-induced vasodilation was further potentiated by CGRP [8-37] (CGRP receptor antagonist, 50 nM), which attenuated the vasodilator response to the CGRP injection. Combined perfusion of AM [22-52] (AM receptor antagonist, 10 to 100 nM) resulted in further inhibition of PNS-induced neurogenic vasodilation without affecting the vasodilator response to the CGRP injection. CGRP [8-37] but not AM [22-52] antagonized vasodilation induced by AM perfusion. These findings suggest that AM presynaptically inhibits neurotransmission of CGRPergic nerves, probably decreasing CGRP release, via receptors different from CGRP receptors.     

In silico and in vitro studies on interaction of novel non-imidazole histamine H3R antagonists with CYP3A4

The paper presents in silico study to explain differences in the influence of the series of non-imidazole histamine receptor H₃ ligands on the activity of cytochrome P-450 3A4 isoform, which was verified in in vitro tests. The compounds appeared to induce broad range of effects – from significant inhibition (-61% reduction of CYP3A4 control activity) to extreme activation (+713% of control activity). Structure-activity relationship for examined compounds was analyzed, with special attention paid to the influence of substituent and the chain length. Docking, molecular dynamics studies, and their statistical analysis allowed to identify those interactions that can be responsible for determination of particular activity type of a compound toward CYP3A4 (activation/inhibition). It resulted in indication of several amino acid residues, which should be carefully analyzed during estimation of compound effects on CYP3A4 activity.     

Pregnane X receptor-dependent and -independent effects of 2-acetylaminofluorene on cytochrome P450 3A23 expression and liver cell proliferation

The arylamide 2-acetylaminofluorene (AAF) is a powerful carcinogen displaying a marked promoting activity, also known to regulate expression of liver detoxifying proteins. In this study we identified CYP3A23, a major inducible cytochrome P-450 (CYP) isoform, as an AAF target in hepatocytes. Indeed, exposure to AAF of primary rat hepatocytes resulted in a marked up-regulation of CYP3A23 expression at both mRNA and protein levels. Using CYP3A23 reporter gene constructs, we further demonstrated that AAF activated the CYP3A23 Direct Repeat 3 (DR3) promoter element interacting with the nuclear pregnane X receptor (PXR). Moreover, the PXR antagonist ecteinascidin-743 fully suppressed AAF-related CYP3A23 induction. Low doses of AAF inhibiting DNA synthesis in hepatocytes however failed to trigger PXR-related CYP3A23 induction and PXR-negative epithelial liver cells remained sensitive to the mito-inhibitory effects of AAF. Such data indicate that AAF up-regulates CYP3A23 through PXR activation but does not require PXR for exerting its carcinogenic promoting properties based on inhibition of cell growth.     

Identification of novel, orally bioavailable spirohydantoin CGRP receptor antagonists

A rapid analogue approach to identification of spirohydantoin-based CGRP antagonists provided novel, low molecular weight leads. Modification of these leads afforded a series of nanomolar benzimidazolinone-based CGRP receptor antagonists. The oral bioavailability of these antagonists was inversely correlated with polar surface area, suggesting that membrane permeability was a key limitation to absorption. Optimization provided compound 12, a potent CGRP receptor antagonist (K_i = 21 nM) with good oral bioavailability in three species.     

The role of TRPV1 in different subtypes of dorsal root ganglion neurons in rat chronic inflammatory nociception induced by complete Freund's adjuvant

Background

The aberrant release of the neurotransmitters, glutamate and calcitonin-gene related peptide (CGRP), from trigeminal neurons has been implicated in migraine. The voltage-gated P/Q-type calcium channel has a critical role in controlling neurotransmitter release and has been linked to Familial Hemiplegic Migraine. Therefore, we examined the importance of voltage-dependent calcium channels in controlling release of glutamate and CGRP from trigeminal ganglion neurons isolated from male and female rats and grown in culture. Serotonergic pathways are likely involved in migraine, as triptans, a class of 5-HT₁ receptor agonists, are effective in the treatment of migraine and their effectiveness may be due to inhibiting neurotransmitter release from trigeminal neurons. We also studied the effect of serotonin receptor activation on release of glutamate and CGRP from trigeminal neurons grown in culture.

Results

P/Q-, N- and L-type channels each mediate a significant fraction of potassium-stimulated release of glutamate and CGRP. We determined that 5-HT significantly inhibits potassium-stimulated release of both glutamate and CGRP. Serotonergic inhibition of both CGRP and glutamate release can be blocked by pertussis toxin and NAS-181, a 5-HT_1B/1D antagonist. Stimulated release of CGRP is unaffected by Y-25130, a 5-HT₃ antagonist and SB 200646, a 5-HT_2B/2C antagonist.

Conclusion

These data suggest that release of both glutamate and CGRP from trigeminal neurons is controlled by calcium channels and modulated by 5-HT signaling in a pertussis-toxin dependent manner and probably via 5-HT₁ receptor signaling. This is the first characterization of glutamate release from trigeminal neurons grown in culture.     

Synthesis, biological evaluation and molecular modelling studies of novel ACD- and ABD-ring steroidomimetics as inhibitors of CYP17

Two novel classes of non-steroidal substrate mimetics were synthesised and examined for their potency as inhibitors of human CYP17. Selected compounds were tested for inhibition of hepatic CYP enzymes 3A4, 1A2, 2C9 and 2C19. The most promising compound 15 showed a good inhibition of the target enzyme (31% and 66% at 0.2 and 2 microM, respectively), and little inhibition of the most important hepatic enzyme CYP3A4 (6% and 19% inhibition at 0.2 and 2 microM, respectively) and the key enzyme of glucocorticoid biosynthesis CYP11B1 (3% and 23% inhibition at 0.2 and 2 microM, respectively). Docking studies revealed that this compound does not assume the same binding mode as steroidal ligands.     

Calcitonin gene-related peptide regulates amino acid absorption across rat jejunum

The calcitonin gene related peptide (CGRP) is widely distributed in the enteric nervous system and gut afferents. Its role in normal digestion and absorption is not characterised. This study is conducted to elucidate whether CGRP regulates amino acid absorption in the small intestine. In in vivo experiments using the single-pass perfusion technique, intravenous infusion of CGRP (250–750 pmol/kg-min) reduced alanine absorption by 35–40%. The effects were completely blocked by the antagonist hCGRP (8–37). Moreover, intravenous infusion of CGRP antagonist blocked the inhibitory effect of intraluminal capsaicin perfusion on alanine absorption. Similarly, intracerebral injection of CGRP decreased alanine absorption, an effect which was reduced by vagotomy. In vitro experiments using isolated jejunal strips showed that CGRP reduced alanine absorption in a dose-dependent manner. At 6 pM, CGRP decreased alanine absorption by 33%. Similarly, CGRP reduced the absorption of proline and taurine by 20 and 11.5%, respectively. Kinetic studies revealed that CGRP reduces alanine influx into intestinal epithelial cells by inhibiting the affinity of the carriers. It is demonstrated that CGRP is involved in the regulation of jejunal amino acid absorption through intrinsic (enteric) and extrinsic (central) neural mechanisms.     

Intermedin 1-53 in central nervous system elevates arterial blood pressure in rats

Intermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family identified from human and other vertebrate tissues. Preprointermedin can generate various mature peptides by proteolytic cleavage. Amino acid sequence analysis showed cleavage sites located between two basic amino acids at Arg93-Arg94 resulting in the production of prepro-IMD(95-147), namely IMD(1-53). The present study was designed to determine the effects of the IMD(1-53) fragment in the central nervous system (CNS) on mean arterial blood pressure and heart rate in normal rats and its possible mechanism. Rats were given doses of adrenomedullin (ADM) or IMD(1-53), intracerebroventricularly or intravenously, respectively, with continuous blood pressure and heart rate monitoring for 45min. Analysis with CGRP receptor antagonist CGRP(8-37), ADM receptor antagonist ADM(22-52), and anti-prepro-IMD antibody showed that 0.1, 0.5, and 1.0 nmol/kg IMD(1-53), caused a dose-dependent elevation in blood pressure, which was more prominent than the increase with equivalent IMD(1-47) or ADM. As well, IMD(1-53) caused a persistent increase in heart rate. The CNS action of IMD(1-53) could be blocked by ADM(22-52), CGRP(8-37), or prepro-IMD antibody. In contrast to the CNS action, intravenous administration of IMD(1-53) induced a depressor effect. These results suggest that IMD(1-53) is an important regulatory factor in mean arterial blood pressure and heart rate through its central and peripheral bioaction.     

The transmembrane domain of receptor-activity-modifying protein 1 is essential for the functional expression of a calcitonin gene-related peptide receptor

Three receptor-activity-modifying proteins (RAMP) define specific interactions between calcitonin (CT) gene-related peptide (CGRP), adrenomedullin (AM) and amylin, and a CT receptor or a CT receptor-like receptor (CRLR). Both form heterodimeric RAMP/receptor complexes at the cell surface. This association represents a novel principle of G protein-coupled receptor function. RAMP1 is transported to the cell surface together with the CRLR or the CT receptor. Here, we have investigated the functional relevance of the short C-terminal intracellular tail QSKRTEGIV and of the single transmembrane domain of human (h) RAMP1 for their interactions with the hCRLR to constitute a CGRP receptor. To this end, hRAMP1 has been sequentially truncated from the C-terminus, and [(125)I]h alpha CGRP/hRAMP1/hCRLR association at the cell surface and cAMP accumulation in response to h alpha CGRP have been examined. With the C-terminal truncation of hRAMP1 by four amino acids wild-type hRAMP1 function was maintained, and the hCRLR was required for the transport of hRAMP1 to the cell surface. Further truncation of hRAMP1 through removal of the remaining five intracellular amino acids revealed CRLR-independent cell surface delivery but otherwise normal hRAMP1 activity. Sequential shortening of the hRAMP1 transmembrane domain resulted in progressively impaired association with the hCRLR and, as a consequence, abolished CGRP receptor function. In conclusion, the intracellular QSKRT sequence adjacent to the transmembrane domain of hRAMP1 provides a signal for intracellular retention. The sequence is unrelated to consensus endoplasmic reticulum retention/retrieval motives and overridden by the presence of the hCRLR. The entire single transmembrane domain of hRAMP1 together with one hydrophilic amino acid residue at its C-terminus is required for the formation of a fully functional CGRP/hRAMP1/hCRLR receptor complex.     

Calcitonin gene-related peptide inhibits interleukin 2 production by murine T lymphocytes.

Evidence from the literature suggests that the nervous and the immune systems closely interact via neuromediators, which affect the immune system, and cytokines, which control nerve cell growth and activity. Calcitonin gene-related peptide (CGRP) is a neuropeptide that has been identified in numerous tissues including immune organs and inhibits the proliferation of spleen cells. We investigated whether CGRP altered the function of T lymphocytes. We present evidence that CGRP induces a dose-dependent cAMP accumulation in interleukin 2-producing TH1 cells and inhibits their production of interleukin 2. These effects are prevented by CGRP8-37, a CGRP antagonist that is missing the first 7 amino acids. This CGRP-mediated inhibition of interleukin 2 production is accompanied by a decrease in interleukin 2 mRNA accumulation. CGRP also inhibits the accumulation of mRNA coding for tumor necrosis factor-alpha and -beta and interferon-gamma. Thus, we have identified one mechanism by which CGRP inhibits the proliferation of spleen cells.