Structure and biological properties of three calcitonin receptor-stimulating peptides, novel members of the calcitonin gene-related peptide family

In this review, we describe the structure and biological properties of calcitonin receptor-stimulating peptide-1 (CRSP-1), CRSP-2 and CRSP-3, the novel members of the CGRP family. CRSP-1, which has been identified in the pig, cow, dog, and horse, is a specific ligand for the calcitonin (CT) receptor, and porcine CRSP-1 elicits a 100-fold greater effect on a recombinant porcine CT receptor than porcine CT, although this peptide has high structural similarity with CGRP. CRSP-1 is expressed and synthesized mainly in the central nervous system (CNS), pituitary and thyroid gland. In an in vivo experiment, bolus administration of CRSP-1 into rats reduced the plasma calcium concentration, but did not alter blood pressure, indicating its action as a CT receptor agonist in the peripheral circulation. In the CNS, CRSP-1 is also deduced to be an endogenous agonist for the CT receptor. CRSP-2 has been identified in the pig and dog, and CRSP-3 has been identified only in the pig. They are expressed and synthesized mainly in the CNS and thyroid gland. However, their endogenous molecular forms, receptors, and biological activity remain unidentified.     

Calcitonin receptor-stimulating peptide: Its evolutionary and functional relationship with calcitonin/calcitonin gene-related peptide based on gene structure

This review focuses on the evolutionary and functional relationship of calcitonin receptor-stimulating peptide (CRSP) with calcitonin (CT)/calcitonin gene-related peptide (CGRP) in mammals. CRSP shows high sequence identity with CGRP, but distinct biological properties. CRSP genes (CRSPs) have been identified in mammals such as pigs and dogs of the Laurasiatheria, but not in primates and rodents of the Euarchontoglires or in non-placental mammals. CRSPs have genomic organizations highly similar to those of CT/CGRP genes (CT/CGRPs), which are located along with CGRPs in a locus between CYP2R1 and INSC, while the other members of the CGRP superfamily, adrenomedullin and amylin, show genomic organizations and locations distinct from CT, CGRP, and CRSP. Thus, we categorized these three peptides into the CT/CGRP/CRSP family. Non-placental mammals having one and placental mammals having multiple CT/CGRP/CRSP family genes suggests that multiplicity of CT/CGRP started at an early stage of mammalian evolution. In the placental mammals, Laurasiatheria generally possesses multiple CRSPs and only one CT/CGRP, while Euarchontoglires possesses CT/CGRP and CGRPβ but no CRSP, indicating an increase in the diversity and multiplicity of this family of genes in mammalian evolution. Phylogenetic analysis suggests that some CRSPs have been generated very recently in mammalian evolution. Taken together, the increase in the number and complexity of the CT/CGRP/CRSP family genes may have due to evolutionary pressure to facilitate adaptation during mammalian evolution. In this regard, it is important to elucidate the physiological roles of CT, CGRP and CRSP from the viewpoint of the CT/CGRP/CRSP family even in Euarchontoglires.     

Identification, structural determination, and biological activity of bovine and canine calcitonin receptor-stimulating peptides

We have recently identified in porcine brain a series of new peptides, designated calcitonin receptor-stimulating peptide-1 (CRSP-1), CRSP-2, and CRSP-3, but failed to find their counterparts in humans and rodents by either database searching or experimental cross-hybridization. In this study, we isolated cDNAs encoding precursors of bovine CRSP-1, canine CRSP-1, and canine CRSP-2 from their thyroid cDNA libraries. Although the deduced mature amino acid sequences of bovine and canine CRSP-1s and canine CRSP-2 showed identity with their respective porcine CRSP counterparts, none of them had a C-terminal amide structure. In LLC-PK(1) cells endogenously expressing the calcitonin (CT) receptor, bovine and canine CRSP-1s enhanced the cAMP production, while canine CRSP-2 did not stimulate it at all. Equine CGRP-I had a high identity in its amino acid sequence with porcine CRSP-1 and stimulated LLC-PK(1) cells at a potency comparable to that of porcine CT. None of these CRSPs or equine CGRP-I stimulated the CT-like receptor, even in the presence of receptor activity-modifying proteins. These results demonstrate that CRSP-1, a new class of biologically active peptide, is present in animals evolutionarily close to pigs and induces its activity through the calcitonin receptor, suggesting a wide existence and common properties of this peptide in mammals.     

Comparisons between the effects of calcitonin receptor-stimulating peptide and intermedin and other peptides in the calcitonin family on bone resorption and osteoclastogenesis

Calcitonin receptor-stimulating peptide (CRSP) and intermedin (IMD) are two recently discovered peptides in the calcitonin (CT) family of peptides. CRSP and IMD, similar to CT, calcitonin gene-related peptide (CGRP), and amylin (AMY), but in contrast to adrenomedullin (ADM), inhibited bone resorption in mouse calvarial bones. CRSP and IMD, similar to CT, CGRP, AMY, but in contrast to ADM, decreased formation of osteoclasts and number of pits in bone marrow macrophage cultures stimulated by M-CSF and RANKL, with no effect on the expression of a number of genes associated with osteoclast progenitor cell differentiation. CRSP and IMD inhibited osteoclastogenesis at a late stage but had no effect on DC-STAMP mRNA. IMD, similar to CGRP, AMY, and ADM stimulated cyclic AMP formation in M-CSF expanded osteoclast progenitor cells lacking CT receptors (CTRs). RANKL induced CTRs and a cyclic AMP response also to CT and CRSP, and increased the cyclic AMP response to CGRP, AMY, and IMD but decreased the response to ADM. Our data demonstrates that CRSP and IMD share several functional properties of peptides in the CT family of peptides, including inhibition of bone resorption and osteoclast formation. The data also show that the reason why ADM does not inhibit osteoclast activity or formation is related to the fact that RANKL decreases ADM receptor signaling through the adenylate cyclase-cyclic AMP pathway. Finally, the findings indicate that activation by CGRP, AMY, and IMD may include activation of both CT and CT receptor-like receptors.     

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.     

降钙素基因相关肽与偏头痛关系的研究

偏头痛是临床上常见的多发性疾病,主要表现为一侧或双侧头部反复发作的搏动性疼痛,可伴有恶心、呕吐、视物异常等神经症状。关于偏头痛的发病机制研究尚无定论。偏头痛的发病机理一直受到人们的关注,近年来一些研究表明,偏头痛的发作与头颅血管周围组织产生的血管活性物质,特别是与三叉神经血管系统的降钙素基因相关肽(calcitonin gene-relatedpeptide,CGRP)的含量变化关系密切。     

Biological actions of human and rat calcitonin and calcitonin gene-related peptide

We assessed the central and peripheral biological actions of human and rat calcitonin and calcitonin gene-related peptide (CGRP). After intravenous administration, human and rat calcitonin, but neither human nor rat CGRP significantly decreased plasma calcium and phosphorus concentrations in awake, freely moving rats. After intracerebroventricular as well as after intravenous administration, human and rat calcitonin and human and rat CGRP significantly inhibited gastric acid secretion in conscious rats. Intracerebroventricular administration of rat calcitonin did not alter plasma calcium and phosphorus concentrations. Linear, partially protected CGRP and calcitonin did not exhibit any biological effects. These studies indicate that calcitonin, but not CGRP, affects calcium and phosphorus homeostasis while both peptides decrease gastric acid secretion similarly. Furthermore, these studies support the hypothesis that the calcium and phosphorus lowering effects of calcitonin are peripheral while the gastric inhibiting actions of the calcitonin and CGRP are mediated by the central nervous system.     

Ontogeny of calcitonin gene-related peptide and calcitonin in the rat thyroid

In this immunohistochemical study, the ontogenic development of calcitonin-gene-related peptide (CGRP) in the rat thyroid was investigated and compared with that of calcitonin using the indirect-immunofluorescence method. Parafollicular cells with immunoreactivity to both CGRP and calcitonin first appeared at an early stage of gestation (days 17 and 18) in the central portion of the thyroid. Cells immunoreactive to CGRP and calcitonin had became numerous by gestational day 22. After postnatal day 7, CGRP- and calcitonin-immunoreactive (C-IR) cells increased rapidly both in number and in the intensity of their fluorescence. In 14- to 90-day old rats, many intensely immunoreactive cells were distributed in the central portion of the thyroid. The cells immunoreactive to CGRP and to calcitonin had an almost identical ontogenic appearance. In 14-day-old and adult rats, C-IR cells also exhibited CGRP immunostaining, suggesting that these cells simultaneously produce and store CGRP during ontogeny.     

Ontogeny of calcitonin gene-related peptide and calcitonin in the rat thyroid

Summary In this immunohistochemical study, the ontogenic development of calcitonin-gene-related peptide (CGRP) in the rat thyroid was investigated and compared with that of calcitonin using the indirect-immunofluorescence method. Parafollicular cells with immunoreactivity to both CGRP and calcitonin first appeared at an early stage of gestation (days 17 and 18) in the central portion of the thyroid. Cells immunoreactive to CGRP and calcitonin had became numerous by gestational day 22. After postnatal day 7, CGRP- and calcitonin-immunoreactive (CIR) cells increased rapidly both in number and in the intensity of their fluorescence. In 14- to 90-day old rats, many intensely immunoreactive cells were distributed in the central portion of the thyroid. The cells immunoreactive to CGRP and to calcitonin had an almost identical ontogenic appearance. In 14-day-old and adult rats, C-IR cells also exhibited CGRP immunostaining, suggesting that these cells simultaneously produce and store CGRP during ontogeny.     

Functional expression of heteromeric calcitonin gene-related peptide and adrenomedullin receptors in yeast.

The ability of G protein-coupled receptors (GPCRs) to form homo- and heteromeric complexes has important implications for the regulation of cellular events. A notable example of heteromer formation is the interaction of the calcitonin receptor-like receptor (CRLR) with different members of the receptor activity modifying protein (RAMP) family, which results in the formation of two different receptors, a calcitonin gene-related peptide (CGRP) receptor and an adrenomedullin receptor. To analyze the role of RAMPs in determining ligand specificity, we have co-expressed CRLR and RAMP proteins in the yeast Saccharomyces cerevisiae, which provides a null system to study the function of mammalian receptors. Co-expression of RAMP1 and CRLR reconstituted a CGRP receptor that was able to activate the pheromone-signaling pathway with pharmacological properties similar to those observed previously in mammalian cells. Co-expression of CRLR with RAMP2 or RAMP3 resulted in a response with the pharmacological properties of an adrenomedullin receptor. These data indicate that RAMPs are necessary and sufficient to determine ligand specificity of CRLR. Contrary to observations in mammalian cells, the glycosylation of CRLR was not affected by the presence of RAMPs in yeast, indicating that glycosylation of CRLR is not the prime determinant of ligand specificity. The first functional reconstitution of a heteromeric seven transmembrane receptor in yeast suggests this organism as a useful research tool to study the molecular nature of other heteromeric receptors.     

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.     

Stimulation of calcitonin secretion in the pig by calcitonin gene-related peptide

Pig thyroid glands were surgically isolated in situ and perfused with autologous blood to which was added known concentrations of calcitonin gene-related peptide (αCGRP). When thyroids were perfused with measured concentrations of CGRP within the range of 0.6–600 nM, the secretion rate of calcitonin (CT) was stimulated while the release of T₃, T₄, and somatostatin remained unchanged. Specific binding of ¹²⁵I-CGRP to pig thyroid plasma membranes was demonstrated, and binding was inhibited by unlabelled CGRP but not by CT or by other peptides unrelated structurally to CGRP. The findings indicate that the pig thyroid gland contains plasma membrane binding sites for CGRP and that CGRP is capable of stimulating the secretion of CT.     

Potent anti-inflammatory action of calcitonin gene-related peptide.

Calcitonin gene-related peptide (CGRP), but not substance P (SP), was found to inhibit edema-promoting actions of inflammatory mediators (histamine, leukotrine B4, 5-hydroxytryptamine) in vivo in the hamster cheek pouch, human skin, and rat paw. The effect of CGRP was present in the low nanomolar dose range, and it was mimicked by activation of sensory nerves with capsaicin which caused release of endogenous CGRP-like immunoreactivity (IR). The findings provide new information on the potential impact of sensory nerve activation during inflammatory processes by indicating that sensory nerves may play an anti-inflammatory role.     

Activities of calcitonin gene-related peptide (CGRP) and related peptides at the CGRP receptor.

In guinea pig pancreatic acini rat calcitonin gene-related peptide (CGRP) increased amylase release 2-fold, salmon calcitonin had an efficacy of only 44% of that of CGRP and [Tyr0]CGRP(28-37) and human calcitonin had no actions. [Tyr0]CGRP(28-37), but not human calcitonin, antagonized the actions of CGRP in pancreatic acini with an IC50 of 3 microM. [Tyr0]CGRP(28-37) produced a parallel rightward shift in the dose-response curve for CGRP-stimulated amylase secretion. The inhibition was specific for CGRP and was reversible. Studies with 125I-CGRP demonstrated that CGRP, salmon calcitonin and [Tyr0]CGRP, but not human calcitonin, interacted with CGRP receptors on pancreatic acini. These results indicate that various CGRP-related peptides demonstrate different relationships between their abilities to occupy the CGRP receptor and to affect biologic activity, with CGRP itself being a full agonist, salmon calcitonin a partial agonist, [Tyr0]CGRP(28-37) a competitive antagonist, and human calcitonin having no actions.     

降钙素基因相关肽受体组分蛋白

降钙素基因相关肽受体组分蛋白(calcitonin gene-related peptide-receptor component protein,CGRP-RCP)是降钙素基因相关肽受体的一个具有146/148个氨基酸的胞内膜周边蛋白,特异地与降钙素受体样受体(calcitonin receptor-like receptor,CRLR)相互作用并促进CGRP和肾上腺髓质素的信号跨膜转导,现认为CGRP-RCP也是G蛋白偶联受体中一个动态的调节器。CGRP-RCP的mRNA在人和鼠的几乎所有组织均可检测到,在小鼠睾丸中分布尤其明显。在哺乳动物中,CGRP-RCP与C17(酵母菌中聚合酶III的必需亚基)是直系同源蛋白,人体的CGRP-RCP能取代酵母中的C17,发挥与C17相同的生物学作用。