Calcitonin Receptor-stimulating Peptide, a New Member of the Calcitonin Gene-related Peptide Family

Abstract

We isolated a novel biologically active peptide, designated calcitonin receptor-stimulating peptide (CRSP), from the acid extract of the porcine brain by monitoring cAMP production in the porcine kidney cell line LLC-PK 1 . Determination of the amino acid sequence and cDNA analysis encoding a CRSP precursor showed that this peptide has ϳ60% identity in the amino acid sequence with human calcitonin gene-related peptide type-␣ (␣CGRP), type-␤ (␤CGRP), and porcine CGRP. Northern blot analysis and radioimmunoassay demonstrated that CRSP is expressed mainly in the thyroid gland and the central nervous system, in which the calcitonin receptor was abundantly expressed. Synthetic CRSP elicited a potent stimulatory effect on the cAMP production in LLC-PK 1 cells. Although it shows significant sequence similarity with CGRPs, this peptide did not elicit cAMP elevation in cells that endogenously expressed a CGRP receptor or an adrenomedullin receptor or were transfected with either of these recombinant receptors. Administration of CRSP into anesthetized rats did not alter the blood pressure but induced a transient decrease in the plasma calcium concentration. In fact, this peptide potently increased the intracellular cAMP concentration in COS-7 cells that expressed the recombinant calcitonin receptor. These unique properties indicate that CRSP is not a porcine counterpart of ␤CGRP and probably elicits its biological effects via the calcitonin receptor.Isolation and sequence determination of new biologically active peptides have greatly advanced our understanding of the communication between cells and tissues. Various biologically active peptides, such as natriuretic peptides (1-3) and neuromedins (4), were isolated by monitoring smooth muscle contraction and relaxation. This strategy was useful, but relatively large amounts of tissue extracts were required to monitor the biological activities. Another strategy involving the measurement of intracellular second messenger levels has often been adopted in recent purification studies, and two methods have been successful to date. The first method is to monitor the second messenger levels in intact cultured cells obtained from a particular organ. Pituitary adenylate cyclase-activating peptide and adrenomedullin (AM) 1 were identified by monitoring the adenylyl cyclase activity in the anterior pituitary cells (5) and platelets (6), respectively. The second method is to monitor the second messenger levels in cells expressing a particular orphan receptor. Orexins and ghrelin were purified by monitoring a transient intracellular calcium elevation in HEK293 cells expressing OX 1 R or OX 2 R receptor (7) and growth hormone secretagogue receptor (8), respectively.

Identification and biological activity of ovine and caprine calcitonin receptor-stimulating peptides 1 and 2

“…Central administration of porcine CRSP-1 to rats suppressed food intake, increased body temperature, and decreased locomotor activity (Sawada et al 2006). Systemic administration of porcine CRSP-1 in anaesthetized rats induced no significant change in arterial pressure but did induce a significant reduction in plasma Ca (Katafuchi et al 2003a). There have been no in vivo studies of the biological actions of a CRSP-1 peptide administered to the species of origin.…”

“…We have recently reported isolation of a novel peptide, calcitonin (CT) receptor-stimulating peptide (CRSP-1), from porcine brain extracts by monitoring cAMP release from the kidney epithelial cell line LLC-PK 1 (Katafuchi et al 2003a). We have since identified two additional closely related peptides CRSP-2 and CRSP-3 also from porcine brain (Katafuchi et al 2003b) and have also identified homologous peptides from cattle, dogs, and horse (Katafuchi et al 2004).…”

“…Each of these peptides shows sequence homology to CT and CT gene-related peptide (CGRP) and thus they make up a part of the CGRP family. Both mRNA expression and immunoreactive peptide content for CRSP-1 is widely expressed throughout the central nervous system and periphery with particularly high levels observed in the midbrain, hypothalamus, pituitary, and thyroid (Katafuchi et al 2003a). CRSP-1 is a specific ligand for the CT receptor (CT-R) with porcine CRSP-1 evoking a 100-fold greater stimulation of cAMP from recombinant CT-R than porcine CT itself (Katafuchi et al 2003a).…”

“…CRSP-1 is a specific ligand for the CT receptor (CT-R) with porcine CRSP-1 evoking a 100-fold greater stimulation of cAMP from recombinant CT-R than porcine CT itself (Katafuchi et al 2003a). By contrast, despite significant sequence homology to CGRP and adrenomedullin (AM), another member of the CGRP family, the CRSPs, appear not to be ligands for any of the combinations of the CT-like receptor (CL-R) and the three receptor activitymodifying proteins (RAMP1-3), namely the CGRP and AM receptors (Katafuchi et al 2003a(Katafuchi et al ,b, 2004.…”

7TM Receptors

“…AM2/Intermedin (AM2/IMD) can also activate CGRP1, AM1, AM2 and AMY1 receptors, albeit less potently than the cognate agonists (Ogoshi et al, 2003;Roh et al, 2004;Hay et al, 2005). CT receptor-stimulating peptide (CRSP) is another member of the family with selectivity for the CT receptor; it has not been found in humans (Katafuchi et al, 2003). BIBN4096BS is the most selective antagonist available, having a high selectivity for CGRP1 receptors, with a particular preference for those of primate origin.…”