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.     

Diverse functional motifs within the three intracellular loops of the CGRP1 receptor

The CGRP(1) receptor exists as a heterodimeric complex between a single-pass transmembrane accessory protein (RAMP1) and a family B G-protein-coupled receptor (GPCR) called the calcitonin receptor-like receptor (CLR). This study investigated the structural motifs found in the intracellular loops (ICLs) of this receptor. Molecular modeling was used to predict active and inactive conformations of each ICL. Conserved residues were altered to alanine by site-directed mutagenesis. cAMP accumulation, cell-surface expression, agonist affinity, and CGRP-stimulated receptor internalization were characterized. Within ICL1, L147 and particularly R151 were important for coupling to G(s). R151 may interact directly with the G-protein, accessing it following conformational changes involving ICL2 and ICL3. At the proximal end of ICL3, I290 and L294, probably lying on the same face of an alpha helix, formed a G-protein coupling motif. The largest effects on coupling were observed with I290A; additionally, it reduced CGRP affinity and impaired internalization. I290 may interact with TM6 to stabilize the conformation of ICL3, but it could also interact directly with Gs. R314, at the distal end of ICL3, impaired G-protein coupling and to a lesser extent reduced CGRP affinity; it may stabilize the TM6-ICL3 junction by interacting with the polar headgroups of membrane phospholipids. Y215 and L214 in ICL2 are required for cell-surface expression; they form a microdomain with H216 which has the same function. This study reveals similarities between the activation of CLR and other GPCRs in the role of TM6 and ICL3 but shows that other conserved motifs differ in their function.     

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.     

Aspartate(69) of the calcitonin-like receptor is required for its functional expression together with receptor-activity-modifying proteins 1 and -2

The calcitonin-like receptor (CLR) associated with receptor-activity-modifying proteins (RAMP) 1 or -2 recognizes calcitonin gene-related peptide (CGRP) and adrenomedullin (AM), respectively. The amino acid sequence CNRTWDGWLCW corresponding to residues 64-74 in the extracellular N-terminus of the CLR is conserved. The Asp(69) (D(69)) is present in all family B1 G-protein-coupled receptors. Here the D(69) of a V5-tagged mouse CLR has been mutated to Ala (A), Glu (E), and Asn (N). The function of the intact and the mutant CLR was investigated in COS-7 cells coexpressing myc-tagged mouse RAMP1 or -2. In CLR/RAMP1 and -2 expressing cells CGRP and AM stimulated cAMP formation with an EC(50) of 0.17 and 0.50 nM, respectively. The expression of the D69A, D69E, and D69N mutants at the cell surface was comparable to that of the intact CLR. cAMP stimulation by CGRP and AM was abolished in the D69A mutant. With the D69E mutant the EC(50) of CGRP and AM were 1000-fold higher than those with the intact CLR. With the D69N mutant the EC(50) of CGRP was 0.48 nM and that of AM 0.44 nM, but the maximal cAMP formation was reduced to 24% and to 12% of cells with the intact CLR. Co-immunoprecipitation of RAMP1 with the CLR, indicating complex formation, was reduced with the D69A, D69N, and D69E mutants. RAMP2 co-precipitated with the mutant receptors indistinguishable from the intact CLR. In conclusion, mutation of D69 to N, E or A in the CLR did not affect its expression at the cell surface, but impaired or abolished the CGRP and AM receptor function in the presence of RAMP1 and -2, respectively.     

Evidence for Conservation of the Calcitonin Superfamily and Activity-regulating Mechanisms in the Basal Chordate Branchiostoma floridae: INSIGHTS INTO THE MOLECULAR AND FUNCTIONAL EVOLUTION IN CHORDATES*

The calcitonin (CT)/CT gene-related peptide (CGRP) family is conserved in vertebrates. The activities of this peptide family are regulated by a combination of two receptors, namely the calcitonin receptor (CTR) and the CTR-like receptor (CLR), and three receptor activity-modifying proteins (RAMPs). Furthermore, RAMPs act as escort proteins by translocating CLR to the cell membrane. Recently, CT/CGRP family peptides have been identified or inferred in several invertebrates. However, the molecular characteristics and relevant functions of the CTR/CLR and RAMPs in invertebrates remain unclear. In this study, we identified three CT/CGRP family peptides (Bf-CTFPs), one CTR/CLR-like receptor (Bf-CTFP-R), and three RAMP-like proteins (Bf-RAMP-LPs) in the basal chordate amphioxus (Branchiostoma floridae). The Bf-CTFPs were shown to possess an N-terminal circular region typical of the CT/CGRP family and a C-terminal Pro-NH₂. The Bf-CTFP genes were expressed in the central nervous system and in endocrine cells of the midgut, indicating that Bf-CTFPs serve as brain and/or gut peptides. Cell surface expression of the Bf-CTFP-R was enhanced by co-expression with each Bf-RAMP-LP. Furthermore, Bf-CTFPs activated Bf-CTFP-R·Bf-RAMP-LP complexes, resulting in cAMP accumulation. These results confirmed that Bf-RAMP-LPs, like vertebrate RAMPs, are prerequisites for the function and translocation of the Bf-CTFP-R. The relative potencies of the three peptides at each receptor were similar. Bf-CTFP2 was a potent ligand at all receptors in cAMP assays. Bf-RAMP-LP effects on ligand potency order were distinct to vertebrate CGRP/adrenomedullin/amylin receptors. To the best of our knowledge, this is the first molecular and functional characterization of an authentic invertebrate CT/CGRP family receptor and RAMPs.     

Structure-function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors

The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM₁ and AM₂, respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM₂/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM₂ potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM₂ and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM₂ and CGRP receptors.     

Molecular and functional characterization of adrenomedullin receptors in pufferfish

The receptors for the calcitonin gene-related peptide (CGRP)/adrenomedullin (AM) family peptides were characterized in the mefugu Takifugu obscurus, a euryhaline fugu species very close to Takifugu rubripes, which has as many as five adrenomedullin genes (AM1-5). CGRP and AM share a G protein-coupled core receptor called calcitonin receptor-like receptor (CLR), and the specificity of the CLR is determined by the interaction with receptor activity-modifying proteins (RAMPs). Through database mining, three CLRs (CLR1-3) and five RAMPs (RAMP1-5) were identified, and all of them were cloned by RT-PCR and characterized by functional expression in COS7 cells in every possible combination of CLR-RAMP. The following combinations generated cAMP in response to physiological concentrations of CGRP, AM1 (an ortholog of mammalian AM), AM2, and AM5: CLR1-RAMP1/4 (CGRP), CLR1-RAMP2/3/5 (AM1), CLR2-RAMP2 (AM1), CLR1-RAMP3 (AM2), and CLR1-RAMP3 (AM5). Their expressions were found by Northern blot analysis to be tissue specific and salinity dependent. For example, CLR1-RAMP5 and CLR1-RAMP2 are expressed specifically in the gill and kidney, respectively, suggesting their involvement in osmoregulation. Furthermore, relatively high levels of CLRs and RAMPs were found in the spleen and ovary, suggesting roles in the immune and female reproductive systems. Immunohistochemistry revealed that AM receptors of the following types are expressed in the locations, indicated in brackets, of the mefugu gill and kidney: CLR1-RAMP5 (interlamellar vessels), CLR2-RAMP2 (pillar cells), and CLR1-RAMP2 (apical side of renal proximal tubule cells).     

Expression of the calcitonin receptor, calcitonin receptor-like receptor, and receptor activity modifying proteins during osteoclast differentiation

The expressions of the calcitonin receptor (CTR), the calcitonin receptor-like receptor (CLR), the receptor activity-modifying proteins (RAMP) 1-3, and of the receptor component protein (RCP) have been studied in mouse bone marrow macrophages (BMM) during osteoclast differentiation, induced by treatment with M-CSF and RANKL. Analyses of mRNA showed that CLR and RAMP1-3, but not CTR, were expressed in M-CSF stimulated BMM. RANKL gradually increased CTR mRNA, transiently enhanced CLR and transiently decreased RAMP1 mRNA, but did not affect RAMP2, RAMP3, or RCP mRNA. However, RANKL did not affect protein levels of CLR or RAMP1-3 as assessed by Western blots or FACS analyses, whereas immunocytochemistry showed enhanced CTR protein. Analyses of cAMP production showed that BMM cells expressed functional receptors for calcitonin gene-related peptide (CGRP), amylin, adrenomedullin, and intermedin, but not for calcitonin and calcitonin receptor stimulating peptide (CRSP), but that RANKL induced the expression of receptors for calcitonin and CRSP as well. Calcitonin, CGRP, amylin, adrenomedullin, intermedin, and CRSP all down regulated the CTR mRNA, but none of the peptides caused any effects on the expression of CLR or any of the RAMPs. Our data show that BMM cells express receptors for CGRP, amylin, adrenomedullin, and intermedin and that RANKL induces the formation of receptors for calcitonin and CRSP in these cells. We also show, for the first time, that the CTR is not only down regulated by signaling through the CTR but also by the peptides signaling through CLR/RAMPs.     

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.     

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.     

Localization of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) in human gastrointestinal tract

Calcitonin gene-related peptide (CGRP) exerts its diverse effects on vasodilation, nociception, secretion, and motor function through a heterodimeric receptor comprising of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). Despite the importance of CLR·RAMP1 in human disease, little is known about its distribution in the human gastrointestinal (GI) tract, where it participates in inflammation and pain. In this study, we determined that CLR and RAMP1 mRNAs are expressed in normal human stomach, ileum and colon by RT-PCR. We next characterized antibodies that we generated to rat CLR and RAMP1 in transfected HEK cells. Having characterized these antibodies in vitro, we then localized CLR-, RAMP1-, CGRP- and intermedin-immunoreactivity (IMD-IR) in various human GI segments. In the stomach, nerve bundles in the myenteric plexus and nerve fibers throughout the circular and longitudinal muscle had prominent CLR-IR. In the proximal colon and ileum, CLR was found in nerve varicosities of the myenteric plexus and surrounding submucosal neurons. Interestingly, CGRP expressing fibers did not co-localize, but were in close proximity to CLR. However, CLR and RAMP1, the two subunits of a functional CGRP receptor were clearly localized in myenteric plexus, where they may form functional cell-surface receptors. IMD, another member of calcitonin peptide family was also found in close proximity to CLR, and like CGRP, did not co-localize with either CLR or RAMP1 receptors. Thus, CGRP and IMD appear to be released locally, where they can mediate their effect on their receptors regulating diverse functions such as inflammation, pain and motility.     

Pharmacology of the human CGRP1 receptor in Cos 7 cells

Only limited pharmacological characterization of the CGRP1 receptor, a heterodimer of the calcitonin (CT) receptor-like receptor (CL) and receptor activity-modifying protein 1 has been performed in cells that do not endogenously express RAMP2. We characterized the receptor in RAMP-deficient Cos 7 cells by measuring cAMP responses following agonist treatment in the absence or presence of antagonists. Potent cAMP responses to human alpha-and beta-CGRP (Cys(Et)2,7)halphaCGRP and human adrenomedullin (AM) were observed. Adrenomedullin15-52 was also an effective agonist of the CGRP1 receptor but human and salmon calcitonin and rat amylin were only weak agonists. As expected, BIBN4096BS and CGRP(8-37) were effective antagonists of the CGRP1 receptor. (Cys(Acm)2,7)halphaCGRP also antagonized CGRP responses. Antagonists of related receptors were only weakly able to inhibit CGRP responses.     

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.     

Identification of the human receptor activity-modifying protein 1 domains responsible for agonist binding specificity

When co-expressed with receptor activity-modifying protein (RAMP) 1, calcitonin receptor-like receptor (CRLR) can function as a receptor for both calcitonin gene-related peptide (CGRP) and adrenomedullin (AM). To investigate the structural determinants of ligand binding specificity, we examined the extracellular domain of human (h) RAMP1 using various deletion mutants. Co-expression of the hRAMP1 mutants with hCRLR in HEK-293 cells revealed that deletion of residues 91-94, 96-100, or 101-103 blocked [125I]CGRP binding and completely abolished intracellular cAMP accumulation normally elicited by CGRP or AM. On the other hand, the deletion of residues 78-80 or 88-90 significantly attenuated only AM-evoked responses. In all of these cases, the receptor heterodimers were fully expressed at the cell surface. Substituting alanine for residues 91-103 one at a time had little effect on CGRP-induced responses, indicating that although this segment is essential for high affinity agonist binding to the receptors, none of the residues directly interacts with either CGRP or AM. This finding suggests that RAMPs probably determine ligand specificity by contributing to the structure of the ligand-binding pocket or by allosteric modulation of the conformation of the receptor. Interestingly, the L94A mutant up-regulated surface expression of the receptor heterodimer to a greater degree than wild-type hRAMP1, thereby increasing CGRP binding and signaling. L94A also significantly increased cell surface expression of the hRAMP1 deletion mutant D101-103 when co-transfected with hCRLR, and expression of a L94A/D101-103 double mutant markedly attenuated the activity of endogenous RAMP1 in HEK-293T cells.     

Selective inactivation of adrenomedullin over calcitonin gene-related peptide receptor function by the deletion of amino acids 14-20 of the mouse calcitonin-like receptor

The receptors for the neuropeptide calcitonin (CT) gene-related peptide (CGRP) and the multifunctional peptide hormone adrenomedullin (AM) are calcitonin-like receptor (CLR)/receptor-activity-modifying protein (RAMP) 1 and CLR/RAMP2 heterodimers, respectively. Here, the amino acid sequence TRNKIMT, corresponding to the residues 14-20 of the N terminus of the mouse (m) CLR, was found to be required for a functional mCLR/RAMP2 AM receptor. The deletion of amino acids 14-20 (Delta14-20) or their substitution by alanine (14-20A) did not affect the heterodimerization of the mCLR with mRAMP1 or mRAMP2, and the levels of expression at the surface of transiently transfected COS-7 cells were not altered. In mRAMP1/mCLR- or mRAMP1/mCLR-(Delta14-20)-expressing cells CGRP stimulated cAMP formation with EC(50) values of 0.12 +/- 0.01 and 1.5 +/- 0.4 nm, respectively. In mRAMP2/mCLR-expressing cells the EC(50) of AM was 0.8 +/- 0.2 nm. However, in cells expressing mRAMP2/mCLR-(Delta14-20) up to 10(-6) m AM failed to stimulate cAMP production. In mRAMP2/mCLR-(14-20A) expressing cells the cAMP response to AM was minimally restored, and the EC(50) was >100 nm. In conclusion, the deletion of the amino acid sequence TRNKIMT of the extreme N terminus of the mCLR maintained CGRP receptor function of mRAMP1/receptor heterodimers, but AM no longer activated the mutant mCLR-(Delta14-20) in the presence of mRAMP2. The TRNKIMT sequence is required for normal mCLR/mRAMP2 association, and as a consequence, high affinity AM binding signaling the activation of adenylyl cyclase.     

Activation of Calcitonin Receptor and Calcitonin Receptor-like Receptor by Membrane-anchored Ligands

G protein-coupled receptors (GPCRs) are the most important pharmaceutical targets, and more than 40% of drugs in use today modulate GPCR signaling. A major hurdle in the development of therapies targeting GPCRs is the drug candidate''s nonselective actions in multiple tissues. The ability to spatially control GPCR signaling would provide a venue for developing therapies that require targeted GPCR signaling. Here, we show that the fusion of a RAMP1 co-receptor with the calcitonin gene-related peptide (CGRP), or calcitonin, transforms the RAMP1 from a co-receptor to bona fide membrane-anchored ligands (CGRP-RAMP1 and CAL-RAMP1). The CAL-RAMP1 selectively activates the calcitonin receptor (CR), whereas, the CGRP-RAMP1 activates both the calcitonin receptor-like receptor (CLR) and CR. Unlike a free peptide, which moves freely in the extracellular space and differentiates targets based on molecular affinity, the anchored CGRP-RAMP1 and CAL-RAMP1 ligands confine their activities to individual cells. In addition, our study showed that a CGRP8–37-RAMP1 chimera, but not RAMP1, functions as an antagonist for CGRP-RAMP1-mediated signaling, suggesting that the activation of CLR by CGRP-RAMP1 shares similar molecular mechanisms with the CGRP-mediated activation of CLR/RAMP1 receptor complexes. Taken together, our finding thus provides a novel class of ligands that activate CR and CLR exclusively in an autocrine manner and a proof-of-concept demonstration for future development of targeted therapies aimed at these receptors in specific cell populations.     

Mapping the functional topography of a receptor.

Photoactivatable derivatives of the alpha-neurotoxin II from Naja naja oxiana are useful tools for investigating the three dimensional architecture of the extra-membrane part of the nicotinic acetylcholine receptor from the electric tissue of Torpedo californica. Three derivatives, carrying an azidobenzoyl group in position Lys-15, Lys-26, and Lys-46, respectively, are shown to react differently within the receptor's quaternary structure. Especially the Lys-26 and Lys-46 derivatives can be used for differentiating between the two nonequivalent alpha-subunits. The Lys-26 derivative is applied for probing the receptor subunits next to the alpha-subunit: the gamma-subunit is shown to be located next to the alpha-subunit binding d-tubocurarine with high affinity. The delta-subunit is the neighbor of the low affinity alpha-subunit. We radioiodinated the toxin derivatives and localized the 125I at the His-31 residue of the toxin. Very little label was found in position Tyr-24, the only tyrosine residue of the toxin, or in position His-4, the only other histidine residue. This result is important for the cleavage experiments necessary in attempts to identify the receptor sequence which reacted with the photolabel.     

Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complexes

Calcitonin, calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), and amylin belong to a unique group of peptide hormones important for homeostasis in diverse tissues. Calcitonin is essential for calcium balance, whereas CGRP and ADM are important for neurotransmission and cardiovascular and respiratory regulation. Based on phylogenetic analysis, we identified intermedin as a novel member of the calcitonin/CGRP peptide family. Analysis of intermedin expression indicated that intermedin is expressed primarily in the pituitary and gastrointestinal tract. Intermedin increased cAMP production in SK-N-MC and L6 cells expressing endogenous CGRP receptors and competed with labeled CGRP for binding to its receptors in these cells. In addition, treatment of 293T cells expressing recombinant calcitonin receptor-like receptor (CRLR) and one of the three receptor activity-modifying proteins (RAMPs) showed that a CRLR/RAMP receptor complex is required for intermedin signaling. In contrast to CGRP and ADM, which exhibited a preferential stimulation of CRLR when co-expressed with RAMP1 and RAMP2 or RAMP3, respectively, intermedin represents a nonselective agonist for the RAMP coreceptors. In vivo studies demonstrated that intermedin treatment led to blood pressure reduction in both normal and spontaneously hypertensive rats via interactions with the CRLR/RAMP receptor complexes. Furthermore, in vivo treatment in mice with intermedin led to suppression of gastric emptying activity and food intake. Thus, identification of intermedin as a novel member of the calcitonin/CGRP peptide family capable of signaling through CRLR/RAMP receptor complexes provides an additional player in the regulation of peripheral tissues by CRLR and will allow development of new therapeutic agents for pathologies associated with diverse vascular and gastrointestinal disorders.     

Adrenomedullin: receptor and signal transduction

Adrenomedullin is a vascular tissue peptide and a member of the calcitonin family of peptides, which includes calcitonin, calcitonin-gene-related peptide (CGRP) and amylin. Its many biological actions are mediated via CGRP type 1 (CGRP(1)) receptors and by specific adrenomedullin receptors. Although the pharmacology of these receptors is distinct, they are both represented in molecular terms by the type II family G-protein-coupled receptor, calcitonin-receptor-like receptor (CRLR). The specificity here is defined by co-expression of receptor-activity-modifying proteins (RAMPs). CGRP(1) receptors are represented by CRLR and RAMP1, and specific adrenomedullin receptors by CRLR and RAMP2 or 3. Here we discuss how CRLR/RAMP2 relates to adrenomedullin binding, pharmacology and pathophysiology, and how chemical cross-linking of receptor-ligand complexes in tissue relates to that in CRLR/RAMP2-expressing cells. CRLR, like other type II family G-protein-coupled receptors, signals via G(s) and adenylate cyclase activation. We demonstrated that adrenomedullin signalling in cell lines expressing specific adrenomedullin receptors followed this expected pattern.     

Characterization of functional calcitonin gene-related peptide receptors on rat lymphocytes.

Calcitonin gene-related peptide (CGRP), a vasoactive neuropeptide present in peripheral neurons, is released at local sites of inflammation. In these studies specific high affinity adenylyl cyclase linked CGRP receptors were characterized on rat lymphocytes. The distribution, affinity, and specificity of CGRP receptors was analyzed by radioligand binding. 125I-[His10]CGRP binding to rat lymphocytes was rapid, reaching equilibrium by 20 to 30 min at 22 degrees C, and dependent on cell concentration. The dissociation constants, Kd, for the CGRP receptor on purified T and B lymphocytes are 0.807 +/- 0.168 nM and 0.387 +/- 0.072 nM and the densities are 774 +/- 387 and 747 +/- 244 binding sites/cell, respectively. Competition binding studies determined that rat CGRP inhibits 125I-[His10]CGRP binding to lymphocytes with the highest affinity (Ki = 0.192 +/- 0.073) followed by human CGRP and the CGRP receptor antagonist CGRP8-37. 125I-[His10]CGRP binding to rat lymphocytes was not inhibited by the neuropeptides substance P, calcitonin, or neuropeptide Y. Lymphocyte CGRP receptor proteins were identified by affinity labeling by using disuccinimidyl suberate to covalently cross-link 125I-[His10]CGRP to its receptor. Specifically labeled CGRP binding proteins visualized by SDS-PAGE analysis had molecular masses of 74.5 and 220 kDa. A third high molecular mass protein band which did not penetrate the gel was also observed. In functional studies, CGRP stimulated a rapid, sustained increase in cAMP with an ED50 of approximately 8 pM. In experiments comparing optimal concentrations of isoproterenol, a beta 2-adrenergic agonist, and CGRP, intracellular cAMP elevation after isoproterenol treatment returned to basal levels by 30 min, whereas cAMP was still elevated at 60 min after CGRP treatment. The response to CGRP was specific in that it could be completely blocked by CGRP8-37. The presence of high affinity functional CGRP receptors on T and B lymphocytes provides evidence for a modulatory role for CGRP in regulating lymphocyte function.