No 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.

No abstract

No abstract