~L 1,; t
`1J Fl •JC
`- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . , .
`
`I
`~3G3
`17
`J• 5
`
`ENDOCRINE
`REVIEWS
`
`Volume 17, Number 5, October 1996
`
`1910
`
`PUBLISHED BIMO THLY BY THE ENDOCRINF. C,()('TFTY
`Endo er in~ r' 1:.• v i e w 5
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`Received on : 10-22-9&
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`

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`

`

`Contents
`
`Vol. 17, No. 5, October 1996
`
`Call for Abstracts for The Endocrine Society
`1977 Annual Meeting
`
`Growth Hormone, the Insulin-Like Growth
`Factor System, and the Kidney
`
`Stella Feld and
`Raimund Hirschberg
`
`Growth Hormone and the Insulin-Like Growth
`Factor System in Myogenesis
`
`James R. Fiorini,
`Daina Z. Ewton, and
`Sharon A. Coolican
`
`Endocrine and Metabolic Disturbances in
`Human Immunodeficiency Virus Infection and
`the Acquired Immune Deficiency Syndrome
`
`Deborah E. Sellmeyer and
`Carl Grunfeld
`
`423
`
`481
`
`518
`
`Calcitonin Gene-Related Peptide and Its
`Receptors: Molecular Genetics, Physiology,
`Pathophysiology, and Therapeutic Potentials
`
`Sunil J. Wimalawansa
`
`533
`
`COVER: Computer-enhanced rendition of rat proximal intestine reacted for calbindin-D9k. Redrawn
`from Figure 2 in the article by S. Christakos et al., "Vitamin D-dependent calcium binding
`proteins: ch emistry, distribu tion, functional considerations, and molecular biology" (Endocrine
`Reviews 1989; 10:3-26). AH covers for Volume 17 are variations of this.
`
`Pharmacia Inc. after SA
`
`INDEX OF ADVERTISERS
`Sandoz Pharmaceutical Corp. C2-2A
`and C/4
`
`

`

`01G3-769X/96/S03.00/0
`Endocrine Reviews
`Copyright e 1996 by The Endocrine Society
`
`Vol. 17, No. 5
`Printed in U.S.A.
`
`Calcitonin Gene-Related Peptide and Its Receptors:
`Molecular Genetics, Physiology, Pathophysiology, and
`Therapeutic Potentials
`
`SUNIL J. WIMALAWANSA
`Department of Internal Medicine, University of Texas Medical Branch at Galveston, Galveston, Texas
`77555-1065
`
`I. Introduction
`II. Discovery of Calcitonin Gene-Related Peptide
`Ill. Molecular Genetics and Structure of the CT/ CGRP,
`Amylin, and Adrenomedullin Genes
`A. Adrenomedullin
`B. Amylin
`C. Calcitonin
`IV. Distribution and Localization
`A. Distribution of i-CGRP
`B. Distribution of CGRP receptors
`V. Age-Related Changes of CGRP and Circadian Rhythm
`VI. Structure-Activity
`VII. Secondary and Tertiary Structures of CGRP
`Vlll. CGRP Receptors
`A. CGRP receptor binding
`B. CGRP receptor subtypes
`C. Isolation, cloning, sequencing, and characterizing
`receptors
`IX. Peptide Measurements
`X. Physiology
`XL Role of Nitric Oxide and Other Second Messengers in
`CGRP-Mediated Actions
`XII. Biological Actions of CGRP
`XIII, Therapeutic Potentials
`A. Therapeutic potentials of CGRP and its agonists
`B. Therapeutic potentials of CGRP antagonists
`XIV. Conclusions and Future Prospects
`
`I. Introduction
`
`THE calcitonin (CT) and cakitonin gene-related pefti~e
`
`(CGRP) are derived from the CT/ CGRP gene, which 1s
`localized in chromosome 11. Alternative splicing of the pri(cid:173)
`mary RNA transcript leads to the translati~n of CG~ ai:id
`CT peptides in a tissue-specific manner. This alternative tis(cid:173)
`sue-specific processing of primary ~~A fro1:1 the a-CT/
`CGRP gene in rats generates two distinct peptides, CT and
`CGRP (1, 2). CGRP is a 37-amino acid neuropeptide e~(cid:173)
`pressed predominantly in the nervous system and CT 1s
`
`Address reprint requests to: Sunil J, Wi~_alawansa, M.D.: Ph.D.,
`MRCPath, FRCP, Divisions of General MediCl.lle and End~cnn~logy,
`Department of Internal Medicine and Pha:macology, Uruvers1ty of
`Texas Medical Branch at Galveston, 301 Uruvers1ty Boulevard, 8.104,
`Medical Research Building, Galveston, Texas 77555-1065.
`
`expressed mainly in the thyroid gland. CGRP receptors,
`widely distributed in the body, are the most potent endog·
`enous vasodilatory peptides that have been discovered. De·
`rived from the C cells of the thyroid gland, CT is the most
`potent peptide inhibitor of osteoclast-mediated bone resorp(cid:173)
`tion and is involved primarily in protecting the skeleton
`during periods of "calcium stress" such as growth, preg·
`nancy, and lactation (3).
`In 1961, Copp and colleagues (4) postulated the exis(cid:173)
`tence of the calcium-lowering peptide CT. Its sequence and
`structure were determined by Neher et al. in 1968 (5).
`Similar to CGRP and amylin, CT is a single-ch ain peptide
`hormone consisting of 32 amino acids. In humans, CT is
`secreted by the parafollicular cells (C cells) of the thyroid
`(6, 7), which are of neural crest origin (8). In addition, a
`minority of CT-producing cells are also in the bronchial
`Kulchitsky (K) cells in the lungs (9). These K cells have
`been shown to be the origin of bronchial carcinoid and
`small cell carcinoma of the lung, both of which secrete CT
`(10, 11). Therefore, even after total thyroidectomy, the
`complete absence of CT from the circulation is unlikely,
`and minute amounts of CT in the circulation may be suf(cid:173)
`ficient to counteract the bone-resorbing effects of other
`hormones such as PTH (12).
`Amylin, a 37-amino add peptide, is encoded by a gene
`located in chromosome 12 (thought to be a duplication of
`chromosome 11 during evolution) and shares a 46% amino
`acid homology with CGRP and 20% with human CT (13-18).
`Amylin is predominant in the /3-cells of the islets of the
`pancreas. Its involvement in the pathogenesis of type II di(cid:173)
`abetes is thought to be by deposition as amyloid within the
`pancreas, leading to /3-cell destruction (12). Another member
`of this peptide family is adrenomedullin (ADM), a 52-amino
`acid vasoactive peptide predominantly located in the adrenal
`tissue (19-21). Not only does adrenomedullin13 .. 52 show 24%
`amino acid homology with CGRP, but its biological activity
`profile in the cardiovascular system is similar to that of CGRP
`albeit less potent (12, 22).
`Previous CGRP-related reviews have concentrated on one
`or more of these areas: molecular genetics, biology, structure
`activity of CGRP, molecular biology, or localization (23-26).
`Several excellent reviews covering the regulation of human
`cardiovascular hemodynamics (27, 28) and clinical applica(cid:173)
`tions of CGRP (29) have been published. There is a growing
`
`533
`
`

`

`534
`
`WIMALAWANSA
`
`Vol. 17, No. 5
`
`interest in using CGRP or its analogs as a therapeutic agent
`in a variety of human and animal disorders. The present
`review is a comprehensive overview of molecular genetics,
`structure-activity, and receptors, with special emphasis on
`clinical potentials, including therapeutic, of this ubiquitous
`neuropeptide.
`
`II. Discovery of Calcitonin Gene-Related Peptide
`The CT gene was cloned first by Jacobs et al. in 1980 (30),
`and further characterization was carried out subsequently
`(31). Discovery of CGRP was made in 1983 by molecular
`cloning of the CT gene by Rosenfeld et al. (1). They observed
`that serially transplanted tumors from a rat medullary thy(cid:173)
`roid carcinoma (MTC) lost the ability to produce CT. This
`spontaneous switch from high to low CT production was
`accompanied by a slight increase in the size of the mRNA
`detected by CT cONA probes (1, 2), which was later deter(cid:173)
`mined to be an "altered mRNA" species derived from the
`CT/CGRP gene (32). This altered mRNA was found to be
`derived from the same RNA precursor as a result of alter(cid:173)
`native RNA processing. The mechanism of regulation of
`CT/CGRP production via alternative RNA splicing remains
`unclear. Although the existence of a splicing enhancement
`factor has been postulated, it has not yet been identified (33).
`The peptide deduced from the cDNA for the alternative
`product was named CGRP (rat a-CGRP), 1 and a similar
`peptide was identified later in humans (h-a-CGRP) (34).
`CGRP is a 37-amino acid neuropeptide (Fig. 1) resulting from
`alternative splicing of the primary RNA transcript of the
`CT/ CGRP gene (1, 2, 32) encoded on the short arm of chro(cid:173)
`mosome 11 p14 qter region between the catalase and PTH
`genes (Table 1) (35-39). Alternative splicing of the primary
`RNA transcript leads to the production of CGRP and CT
`peptides in a tissue-specific manner (12). 1n the central ner(cid:173)
`vous system (CNS), for example, splicing of the a-CT I CGRP
`gene produces CGRP, whereas in the C cells of the thyroid
`gland, CT is predominantly formed (37).
`The existence of a second CGRP gene (/3-CGRP), also lo(cid:173)
`cated on chromosome 11, was predicted from further anal(cid:173)
`ysis of rat and human cONA clones ( 40, 41 ). This second gene
`is thought to be due to gene du plication, and it remains
`unclear which of the two genes appeared first (12). Sequence
`
`changel, in exon 4 of the /3-CGRP gene result in selective
`production of {3-CGRP in all tissues in which the gene is
`expressed. The r-a-CGRP differs from r-{3-CGRP by one
`amino acid, and h-{3-CGRP differs by three amino acids from
`the homologous peptide h-a-CGRP (40-43) (Fig. 1). a- and
`{3-CGRP exhibit nearly identical biological activities. Expres(cid:173)
`sion, significance, and the potential interactions of these two
`forms of CGRP in different tissues are yet to be clarified.
`
`III. Molecular Genetics and Structure of the
`CT/CGRP, Amylin, and Adrenomedullin Genes
`A schematic representation of the human a-CT/ CGRP
`gene structure is illustrated in Fig. 2. The CT /CGRP gene
`consists of six exons. The first three exons are constitutively
`spliced in both mRNAs. Exon I is untranslated, exon Il codes
`for the signal peptide, and exon ill codes for the N-terminal
`propeptide. Calcitonin and CGRP sequences are localized in
`exon IV and exon V, respectively. Exon VI is part of the
`a-CGRP mRNA but is untranslated. The primary transcript
`includes all six exons, and CT or CGRP mRNA is formed
`subsequently (Fig. 2) (41, 42). CGRP-containing mRNA is
`produced by splicing together the first three exons with
`exons V and VI located downstream (40). Exon V encodes
`CGRP, a flanking tetrapeptide, while exon Vl encodes the
`3'-untranslated regions of the CGRP mRNA and polyade(cid:173)
`nylation (poly A) signal (1-3). This mRNA is translated to
`generate pro-CGRP peptide, which is subsequently cleaved
`at paired dibasic amino acids to release the 37-amino acid
`CGRP (Fig. 2) (44). The alternative RNA processing of the
`RNA precursor involves differential recognition of exons Ill
`and TV, which encode CT, a carboxy-terminal flanking pep(cid:173)
`tide, katacalcin, and the 3' -untranslated region of CT mRN A.
`Splicing of exon IU to exon IV with polyadenylation at the
`3'-end of exon IV yields CT mRNA (45).
`Both the a-CT/ CGRP gene and the f3-CGRP gene, located
`in chromosome 11 (Table 1), contain six exons (40-42). The
`organization of the /3-CGRP gene is similar to that of the
`a-CT /CGRP(46) gene (36-42). Exon Tis untranslated, exon
`II contains the sequence of the signal peptide, and exon Ill,
`which is 92% homologous to exon Il of the a-CT I CGRP gene,
`codes for the N-terminal propeptide. Exon IV of the {3-CGRP
`gene is 67% homologous to the same region of the a -CT/
`
`o<-Human CGRP
`
`J3 - Human CGRP
`
`FIG. 1. Amino acid sequences of human a- and /3-CGRP. The difference between two peptides are highlighted.
`
`

`

`October, 1996
`
`CALCITONIN GENE-RELATED PEPTIDE
`
`535
`
`TABLE 1. Chromosomal localization of a-CT/CGRP, /3-CGRP, amylin, adrenomedullin, and insulin
`
`Gene
`o-CGRP/CT
`
`/3-CGRP
`
`Amylin
`Adrenomedullin
`
`Insulin
`
`Chromosomal location
`Chromosome 11 (p13-pl5)
`[between catalase (16cM) and PTH (8cM)]
`Chromosome 11 (pl2-qter-pl4)
`
`Chromosome 12 (12 p 12.3 ql4)
`Chromosome 11
`
`Chromosome 11 (pl3-pter)
`
`Reference
`Amara et al., 1982
`Rosenfeld et al., 1983
`Hoppener et al., 1985
`Amara et al., 1985
`Nishi et al., 1989
`Ishimitsu et al., 1994
`(OMIM #103275)
`Owerbach et al., 1980
`
`Ref. number
`32
`l
`37
`40
`131
`127
`
`39
`
`0::~on O ,,:.ru O N-,:~§:e
`peptide CJ0~~C
`
`CT specific /\
`
`d.
`com mon co mg
`
`poly A
`
`5
`
`~:
`non-co~ing sequenci
`r} .
`CGRP specific V
`
`Primary transcript
`Mature mRNA
`
`polyaden~ ation site
`
`V
`
`COR P
`coding
`
`VI
`
`poli A
`t
`V 3'-CGRP
`
`J non-coding
`
`3'
`
`polyadenylation
`site
`
`~
`
`AAA
`
`common U ivDAAA
`CT mRNA
`P recursor peptides +
`ommon (cid:127) filCT ?a ~KC~
`\
`
`7aa
`
`4aa
`
`COOH
`
`N H 7-
`CT precurso/
`
`Mature peptides
`
`NH2
`
`5aa
`
`5aa
`
`c oRP I I f-cooH
`
`CGRPmRNA •
`llcommon. (cid:127)
`CGRP precursor ! 4 aa
`
`37 amino acids
`32 amino acids
`N H 2~ct&cooH
`CGR P -
`-COOH
`NH 2•
`FIG. 2. A schematic representation of the structural organization of the human a-CT/CG RP gene. Two different mRNAs may be produced from
`the alternative processing of the single primary transcript and use of two polyadenylation sites: one coding for the CT precursor ((cid:143) ) and the
`other for the CGRP precursor (a). Posttranslational modifications include intramolecular disulfide bridge formation at the N terminus and
`amidation of the C-terminal residue. CT, Calcitonin, KC, katacalcin; CGRP, calcitonin gene-related peptide.
`
`21 amino acids
`NH 2~KC~COOH
`
`CGRP gene that gives rise to CT. Within the exon IV of the
`(3-CGRP gene, lack of a poly A signal, therefore, prevents
`alternative splicing. Consequently, transcripts from this gene
`produce only CGRP, and it is thought to be a pseudogene for
`CT (47, 48). Overall, the a-CT/ CGRP and (3-CGRP genes
`share 96% homology in the regions of exons II and III. Exon
`IV coding for (3-CGRP gene shows a 92% homology to the
`corresponding exon V of the a -CT/ CGRP gene. On the other
`hand, the exon V of the (3-CGRP gene, which corresponds to
`the exon VI of the a-CT/ CGRP gene, has only 65% homol(cid:173)
`ogy. In humans, these two genes are also called the CALC I
`and CALC II genes. A third CT/ CGRP-like gene has also
`been identified on chromosome 11 and contains nucleotide
`sequences corresponding to exon II and Ill of a-CT/ CGRP
`and /3-CGRP genes (49). However, since it is not transcribed
`it is a pseudogene and does not contain nucleotide sequences
`homologous to CT and CGRP. The divergent nature of exon
`VI allows the use of sequence-specific probes to study the
`differential expressions of these two genes in various tissues
`
`(50). Complete sequences of human CT, a-CGRP, and
`(3-CGRP precursors have been deduced by cloning and anal(cid:173)
`ysis of recombinant DNA from mRNA extracted from hu(cid:173)
`man MTC (41, 42, 51-53).
`The h-a-CGRP is synthesized as a precursor pre-pro-pep(cid:173)
`tide consisting of 128 amino acids. This pre-pro-protein is
`flanked by peptides at both its amino terminus and on its
`carboxy terminus. The mature, C-terminally amidated CGRP
`is derived by further cleavage of the leader sequence (Fig. 2).
`Similarly, the precursor peptide of (3-CGRP consists of 127
`amino acids and is also cleaved at both ends to produce a
`mature, C-terminally amidated peptide (53, 54). Through
`evolution, both CGRP and its receptors have been well con(cid:173)
`served (12, 55-58), which is consistent with their distribution
`and the postulated functions of CGRP. Transcription of the
`a-CT/ CGRP gene is regulated by a cell-specific enhancer
`about 1 kb upstream of the transcription initiation site (59-
`63). The repressing effect of retinoic acid on a-CT/ CGRP
`mRNA levels in the CA-77 thyroid C cell line seems to be due
`
`(cid:127)
`

`

`536
`
`WIMALA W ANSA
`
`Vol. 17, No. 5
`
`to a reduction of the a-CT/ CGRP promoter activity caused
`by repression of this cell-specific enhancer element (64). The
`specific interactions of dexamethasone, estrogens, progestro(cid:173)
`gens, androgens, and vitamin D and the regulation of the
`a/CT-CGRP gene expression have not been addressed fully.
`The alternative transcription of the a-CT/ CGRP gene in
`the thyroid C cells and neural tissues results in the tissue(cid:173)
`specific production of CT and a-CGRP mRNAs. This alter(cid:173)
`native spbcing seems to be regulated by a differential cellular
`capacity to utibze the CT-specific alternative splicing of the
`CT /CGRP primary transcript, which is mediated by cell(cid:173)
`specific differences in components of the constitutive splic(cid:173)
`ing machinery (65). For example, two different sequence
`elements within exon IV of the a-CT /.CGRP gene are nec(cid:173)
`essary for CT-specific splicing (66). These cis-acting factors
`probably interact with trans-acting factors, which then bind
`specifically to exon IV sequences to promote CT-specific
`splicing. On the other hand, some of these may induce ex(cid:173)
`clusion of exon IV, resulting in the formation of a-CGRP
`mRNA (67, 68). The presence of a 66-kDa protein has been
`demonstrated by UV-cross- linking that binds specifically to
`exon IV of the CT /CGRP gene (69). This protein has been
`demonstrated to be a CT-specific facibtative factor, leading
`to a positive regulation by !rans-acting factors (70). Little data
`have been published on the regulation of /3-CGRP gene. After
`peripheral axotomy, a- and /3-CGRP mRNAs are differen(cid:173)
`tially regulated in rat spinal cords and dorsal root ganglia
`(71). In contrast to the stimulatory effects of dexamethasone
`on a-CT/ CGRP gene, the levels of /3-CGRP m.RNA did not
`change after treatment with dexamethasone (72). However,
`more research is necessary to understand the regulation and
`the significance of the /3-CGRP gene.
`RIAs for CGRP have been established to measure peptide
`concentration in circulation, in cerebrospinal fluid (CSF), and
`in tissue extracts (73-81). In addition to RIA for CGRP, we
`have also developed a highly specific and sensitive radio(cid:173)
`receptor assay (RRA) (82). We have used this RRA: 1) to
`demonstrate subtle changes of plasma CGRP levels under a
`number of physiological conditions including circadian vari(cid:173)
`ations of CGRP in plasma (83); and 2) to show an increase in
`plasma CGRP after the administration of oral calcium sup(cid:173)
`plements in patients with essential hypertension (84); and 3)
`as a new tool for diagnosing MTC and C cell hyperplasia (85).
`Both a- and /3-CGRP are present in body fluids such as
`plasma, CSF, and joint fluid and also in many other tissues
`such as spinal cord, gut, and thyroid tissues in humans (43,
`75-78). Like many other biologically active peptides such as
`PTH-related protein (PTHrP) and endothelin, a number of
`different molecular weight forms of CGRP are present in
`body fluids and tissues. We have proven the presence of
`several molecular weight forms of CGRP and the presence of
`both a- and /3-CGRP in tissues, plasma, and CSF in rats and
`humans (75-80). Most of the irnrnunoreactive CGRP (i(cid:173)
`CGRP) present in the circulation is thought to be derived
`from sensory nerve terminals of small capsaicin-sensitive
`fibers (finely myelinated A delta and unmyelinated C fibers)
`(12, 86, 87). CGRP is likely to have an important role in the
`local regulation of blood flow (12, 28, 88) and in modulating
`neurological function (23-28). The primary amino acid se-
`
`quences of the nine homologous CGRPs are illustrated in
`Table 2 (34, 40-43, 89-94).
`In addition to MTC, the presence of CGRP has been un(cid:173)
`equivocally demonstrated in a number of other cancers.
`These include bronchogenic lung cancer, promyelocytic leu(cid:173)
`kemia, insuJinoma, carcinoid tumors, pheochromocytoma,
`prostatic adenocarcinoma, parathyroid adenoma, and a va(cid:173)
`riety of neoplastic cell lines (50, 54, 95-106). In these tumors,
`however, CGRP concentration is uniformly lower than that
`found in MTC.
`
`A. Adrenomedullin
`
`ADM is a 52-amino acid peptide originally isolated from
`a human adrenal pheochromocytoma using a detection sys(cid:173)
`tem based on its ability to elevate platelet cAMP levels (19,
`107). Sequence analysis of cloned human ADM cDNA
`showed that its precursor is 185 amino acids in length, in(cid:173)
`cluding a putative signal peptide. In addition to this pre(cid:173)
`cursor, the gene encodes a unique 20-amino acid sequence,
`designated proadrenomedulbn N-terminal 20 peptide (pro(cid:173)
`AMN20) (19, 20). The complete cDNA sequences encoding
`pig and rat ADM, located in chromosome 11, have also been
`cloned (19-21). Primary amino acid sequence identities of
`human, porcine, and rat ADM with a- and /3-CGRP, and of
`human, eel, and salmon calcitonin are illustrated in Fig. 3.
`lmmunoreactive ADM has been detected by RIA in many
`tissues, including normal adrenal meduIJa, heart, kidney,
`pancreas, intestine, and plasma (19, 108-111).
`The main physiological effects of ADM seem to be vaso(cid:173)
`dilation including an increase in pulmonary blood flow (19,
`107, 108, 112, 113). Other effects include bronchodilation
`(113), inhibition of drinking behavior (114), and an inhibition
`of angiotensin-induced aldosterone secretion (115). The hy(cid:173)
`potensive activity of ADM is second only to CGRP (22, 116-
`121). As with CGRP, reduction of mean blood pressure after
`intravenous administration of ADM (e.g. dosage of 1 nmol/
`kg) is due to a decrease in peripheral vascular resistance (22,
`117-121). That the cardiovascular activities of ADM are in(cid:173)
`hibited after administration of the CGRP antagonist
`CGRP8 -37 (22, 116) suggests that ADM acts via CGRP recep(cid:173)
`tors in cardiovascular tissues, but it is also possible that it acts
`via its own receptor (12).
`
`B. Amylin
`
`Amylin (islet amyloid polypeptide; diabetes-associated
`peptide) is a 37-amino acid peptide first isolated from an
`insulinoma (13-16). Amylin is a member of the CT /CGRP
`family, having 46% of its amino acid sequence identical with
`human CGRP (h-CGRP), a 15% sequence similarity with
`human calcitonin (h-CT), and a 28% sequence identity with
`salmon CT (s-CT) (Fig. 4). It shares lesser amino acid se(cid:173)
`quence similarity with insulin and insulin-like growth fac(cid:173)
`tors, both of which have their DNA sequences' code located
`in chromosome 11 (12, 17, 18). Two structural features that
`are essential for full agonist activity are conserved between
`CT, CGRP, ADM, and amylin. All four peptides contain two
`N-terminal cysteines that form a disulfide bridge resulting in
`an N-terminal loop and a C-terminal amide (12).
`
`

`

`October, 1996
`
`CALCITONIN GENE-RELATED PEPTIDE
`
`537
`
`TABLE 2. Primary amino acid sequences of nine calcitonin gene-related peptides (CGRP)
`
`AA No
`
`Human
`CGRP-/1
`Ala
`
`Asn
`
`Rat
`CGRP-a
`Ser
`
`Asn
`
`Rat
`CGRP-/3
`Ser
`
`Asn
`
`Porcine
`CGRP
`Ser
`
`Asn
`
`Chicken
`CGRP
`
`Frog
`CGRP
`
`Asn
`
`Asn
`
`Human
`CGRP-a
`Ala
`1
`Cys
`2
`Asp
`3
`Thr
`4
`Ala
`5
`Thr
`6
`Cys
`7
`Val
`8
`Thr
`9
`10
`His
`Arg
`11
`12
`Leu
`13
`Ala
`Gly
`14
`15
`Leu
`Leu
`16
`17
`Ser
`Arg
`18
`Ser
`19
`20
`Gly
`21
`Gly
`Val
`22
`Val
`23
`Lys
`24
`Asn
`25
`26
`Asn
`27
`Phe
`28
`Val
`Pro
`29
`Thr
`30
`Asn
`31
`Val
`32
`Gly
`33
`34
`Ser
`Lys
`35
`Ala
`36
`Phe
`37
`Identical amino acid residues from human a-CGRP are indicated with (-), and variations are specified.
`
`Asp
`Phe
`
`Asp
`Phe
`
`Gly
`
`Met
`Ala
`
`Met
`
`Ser
`
`Asp
`
`Asp
`
`Ser
`
`Asp
`
`Glu
`
`Glu
`
`Rabbit
`CGRP
`Gly
`
`Asn
`
`Bovine
`CGRP
`Ser
`
`Asn
`
`Met
`
`Ser
`
`Ser
`
`Glu
`
`Glu
`
`,o
`Peptide
`JO
`20
`50
`10
`- Y R Q 9 N N N r Q O L R 8 F O C R F O T C T V Q IC L A H Q I Y Q P T D IC D IC D II V A P R 9 IC I S P Q (l Y-HH2
`h- ADM
`S P Q (l Y- HH2
`
`- Y RQSIIIIIIF Q G L R S1 GCR F OT C TVQIILA!IQI Y Q
`
`p-ADII
`
`A C D T A T C V T H R L A G L L S R S (l (l V V It Nfiil F V P T II V G S It A •
`
`r-HH2
`
`8 P Q G Y-IIH2
`
`r - llH2
`
`C G N L S T C N L Cl T Y T~D F N IC -
`
`F H T F P Q T A I Cl V (l A - P• HH2
`
`A C N T A T C V T !I R L A G L L S I\ S G G M V It sl:1 F V P T N V G S It A -
`ICC NT ATC A •{~JR LA II r L V !I 9 S N NP GAIL 9 ST NV GS NT 8
`C 9 II L 9 T C Fl L G II L S Q L B H It
`c s II L s T c r:J L o 1t L s Q o L H 11
`
`r•ADII
`
`• y R Q
`
`h-CGI\P- tt -
`
`h -CGRP-~ -
`
`h -amylin (cid:173)
`
`h-CT
`
`e • CT
`
`a - CT
`
`- L Q T Y P I\ T N V G A G V - P· NB2
`
`t. Q T Y P a T II T aG o T - P- HH2
`
`-
`
`-
`
`1 Fm. 3. Primary amino acid sequence homology of various adrenomedullins (ADM), human a- and /3-CGRP, human amylin, and hum an, eel,
`and salmon calcitonin (h-CT, e-CT, and s-CT). Identical amino acids to the human ADM in the peptide sequences are indicated in boxes.
`
`Amylin has also been isolated from pancreatic amyloid
`deposits of type II diabetic patients (15, 16) and is present in
`normal islet cells of the pancreas (17, 18). Amylin is the major
`component of islet amyloid (a deposition of amyloid protein)
`in the pancreas of patients with non-insulin-dependent (type
`II) diabetes mellitus (14, 15). The gene encoding amylin is
`
`located in the short arm of chromosome 12 of the human
`genome (within the q14 pter region, 12p 12.3) (Table 1) (122,
`123). The amylin gene, localized in chromosome 12, was
`mapped by techniques using human-rodent somatic cell hy(cid:173)
`brids (122-125). Chromosome 12 is thought to be derived
`from evolutionary duplication of chromosome 11, which
`
`

`

`538
`
`WIMALAWANSA
`
`Vol. 17, No. 5
`
`1
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`Hurnan-CGRP
`
`TAT C
`
`R L
`
`K N N F V P T N V G S K A F
`
`Human-Amyl in
`
`KCNTATCATQRLANF
`
`Monkey-Amy
`
`KCNTATCATQRLANF
`
`L v[;j S S N N F G A I L S fsl T N V G S N T Y
`
`L v R s s N N F G El I L s W T N v G s 0 T y
`
`Cat-Amylin
`
`K C N T A T C A T Q R L A N F
`
`L 0R S S N N L G A I L S P T N V G S N T Y
`
`Dog-Amylin
`
`K C N T A T C A T Q R L A N F
`
`L V Rl!]s N NL GAILS PT NV GS NT Y
`
`Rat-Amyl in
`
`KC NT ATC AT QR LAN F L V R s s N N LG p V LMP TN VG s NT y
`
`Mouse-Amylin
`
`Hamster-Amy
`
`KC NT ATC AT QR LAN FL V RS SN NL GP V Ll.:JP TN VG SN TY
`K c N T A T c A T Q R L A N F L v~ sG N N L G P v L s P T N v G s N T Y
`
`Guineapig-Am
`
`K C N T A T C A T Q R L T N F L V R S S H N L G A A L L
`
`G S N T Y
`
`Degu- Amylin KC NT ATC AT QR LT NFL V RS SH NL GAAL P
`
`G S NT Y
`
`FIG. 4 . Amino acid sequence comparison between human CGRP and various amylin peptides. Identical amino acids in the peptide sequences
`are indicated in boxes.
`
`contains both a- and {3-CGRP and insulin (12, 35--41, 123-
`127). This indicates that the amylin gene represents another
`gene duplication, in addition to the {3-CGRP gene. The amy(cid:173)
`lin gene, in accordance with the {3-CGRP gene, only gives rise
`to one peptide product. Construction of a family tree using
`a variety of information, including hydropathy plots and
`sequence divergence, suggests that CGRP and amylin have
`been separated for a considerable time. Amylin has been
`proposed as a causative factor in type II diabetes, and several
`other in vivo and in vitro effects have been reported with this
`peptide (128-155).
`
`C. Calcitonin
`
`Transcription of the CT gene in cell-free systems allowed
`for the selection of CT-coding sequences from cloned cDNA
`libraries. Isolation of CT cDNA clones led to the identifica(cid:173)
`tion of a CT mRNA consisting of - 1000 bases that encodes
`a precursor protein of 136 amino acids in rats and 141 amino
`acids in humans (3, 36). Like CGRP, CT is also synthesized
`as a large precursor protein, containing 141 amino acids (Fig.
`2). This pro-CT peptide is cleaved at dibasic amino acid lin