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Megan Hutcherson March 3, 2014 A Study of the hormone Calcitonin Calcitonin is a hypocalcemic agent, acting on both osteoclasts and renal tubules of the kidney to reduce calcium levels in the blood (Pondel 1). Calcitonin prevents bone reabsorption in osteoclasts by inhibiting osteoclast motility and inducing a gradual retraction of the osteoclasts in the body (Masi, Brandi 4). These actions are completed through the use of the second messenger system; where both calcium and CAMP amplify and carry out this bioregulator’s signal to target molecules in the cell (Norris, Carr 54, 55). In addition, calcitonin has the ability to interfere with osteoclast differentiation and inhibit mononucleated precursors to form multinucleated cells (Masi, Brandi 4). Calcitonin is regulated by calcium levels in the blood and Calcitonin will function to decrease systemic levels of serum calcium when hypercalcemia occurs (Wikipedia 1). In addition, calcitonin will be stimulated through the release of gastrin and pentagastrin (Wikipedia 1). Calcitonin and calcitonin gene related peptide are members of the same peptide family, which consists of 6 members including calcitonin gene related peptide one and two, calcitonin, amylin, adrenomedullin, and intermedlin (Barwell, et. all 2). Calitonin gene related peptide functions as a vasodilator throughout the vascular system and CGRP also has a neuromodulatory role in the nervous system (Masi, Brandi 4). It has been proven that calcitonin not only has receptors in osteoclasts and the kidneys, but in the gastrointestinal and reproduction system as well (Masi, Brandi 5,6,7 ). In humans calcitonin is produced by parafolicular cells (c cells) in the thyroid (Norris, Car 508). Parathyroid cells originate from the ultimobrachial body and this is made from the sixth pharyngeal pouch (Norris, Car 508). The ultimobrachial body will be incorporated into the thyroid gland in humans, but it does exist as a separate structure in some vertebrates (Norris, Car 508). Calcitonin is a 32 amino acid polypeptide hormone with a molecular mass of 3418 Da (Masi, Brandi 2). Calcitonin exists in the shape of an alpha helix and it has one disulfide bridge connecting Cysteines at positions 1 and 7 to produce a 7 amino acid ring structure at the amino terminus (Pondel 2). Please see figure one below for an image of the structure of calcitonin. Calcitonin is amphipathic with hydrophobic regions on LEU 4,9,12,16 and hydrophobic regions on the opposite side of this chain, Cys 7, Lys 11, Glu 15, and lys 18 (Pateu 1). Please see figure two below for an image of these hydrophobic regions. The protein sequence for calcitonin has been determined in many species and it has been noted that this sequence is highly conserved at the N terminal loop region, but there divergence in the rest of the sequence (Pondel 2.) “Fully processed CT and CGRP transcripts share sequence identity in the amino terminal regions but are almost entirely different in the carboxy‐terminal regions” (Pondel 3). Calcitonin gene related peptide is a 37 amino acid polypeptide. Thyroid stimulating hormone exhibits a different structure than both calcitonin and calcitonin gene related peptide. TSH is a category one trophic hormone (including LH and FSH) and it is a heterodimeric cysteine knot glycoprotein (Szkundiski 1). This hormone contains an alpha and beta subunit and it is encoded by genes that are located on chromosome 1 and 6. The alpha and beta subunits contain a central cysteine knot which is composed of three disulfide bonds (Szkundiski 1). The alpha subunit of TSH has a two turn alpha helix and the beta subunit is composed of a beta hair pin loop on one side of the cysteine knot. TSH has 3 asparagine linked carbohydrate chains in total with two carbohydrate chains in the alpha subunit and one located in the beta subunit (Szkundiski 1). The carbohydrate component of this hormone makes up 15 to 30% of the alpha and beta subunits weight (Norris, Carr 111). The alpha subunit for LH, FSH, and TSH is identical and the beta subunit is responsible for the hormones individual properties (Norris, Carr 111). Figure 1 Figure 2 Calcitonin is encoded by a gene named CALC‐1 and this gene is located on chromosome 11 in the human genome (Masi, Brandi 2). CALC‐1 is a member of the calcitonin gene family and this gene family includes CALC‐2, CALC‐3, and CALC‐4 (Masi, Brandi 2). CALC‐1 is the only gene in this gene family which produces calcitonin and this gene can also encode calcitonin gene related peptide one and two (Masi, Brandi 2). Calcitonin and calcitonin gene related peptide production depends on the actions of alternative splicing during post‐transcriptional processing (Pondel 3). Alternative splicing occurs after RNA is produced and during this process introns are removed and then exons are spliced together to form mRNA (Norris, Carr 43). The production of calcitonin gene related peptide/calcitonin depends on the inclusion or exclusion of the exon four (Pondel 3). In parafolicular cells of the thyroid CT/CGRP pre‐mRNA is processed to include exon four (Pondel 3). This is followed by polyadenylation (Synthesis of a poly A tail at the three prime end of RNA) and the transcription of Calcitonin (Pondel 3). Calcitonin gene related peptide is formed through the exclusion of exon four and the inclusion of exon five and six (Pondel 3). Exon 6 is used as a polyadenylation site to produce calcitonin gene related peptide. In addition to mRNA processing of CALC‐1, the production of both these hormones is regulated through the use of transcriptional regulatory elements (Pondel 4). CALC‐1 gene’s promoter contains CPG islands near exon one and 1.5kb after this exon (Pondel 4). CPG islands are unmethylated CPG regions of DNA that have a different chromatin structure from bulk chromatin due to the absence of histone H1. In addition CPG islands contain hyperacetylated histones (H3 and H4) and have nucleosome‐free segments of DNA (Pondel 4). The CPG islands at the end of CALC‐1 gene may affect a promoter’s availability to transcription factors (Pondel 4). Therefore it is important to keep an open chromatin conformation, through the use of hypermethylated DNA, to help transcription factors bind to the CALC1 promoter and activate CT gene expression (Pondel 4). The biosynthesis of Calcitonin follows the same mechanisms involved in the synthesis of protein bioregulators including transcription, alternative splicing, and translation. Calcitonin is initially transcribed from CALC‐1 to form a 136 amino acid preporpeptide (PRECT), which contains a leader sequence at the amino terminal region (Pondel 3). This leader sequence is cleaved when PRECT is transported to the endoplasmic reticulum (Pondel 3). After transportation to the endoplasmic reticulum, procalcitonin is produced (PROCT) and transported to the golgi apparatus via exocytosis (Masi, Brandi 2). In the Golgi apparatus PROCT is cleaved by Prohormone Convertase enzymes to produce immature calcitonin (ICT) (Masi, Brandi 2). ICT will then undergo additional protolithic cleavage in secretory vesicles, of the Golgi apparatus, to produce mature calcitonin (MCT) (Masi, Brandi 2). Secretory vesicles will then fuse with a cells plasma membrane and calcitonin will be stored in the cytoplasm till use. Chromogranin B may act as a helper protein to aid with trans‐golgi sorting during biosynthesis (Masi, Brandi 2). Please see figure three bellow for a description of Calcitonin’s biosynthetic pathway. Figure 3 In order for Calcitonin to reduce bone reabsorption and increase calcium excretion from the kidneys it needs to be able to bind to the Calcitonin receptor. The calcitonin receptor is a G Protein coupled receptor and these receptors are often called seven transmembrane receptors since they pass through the plasma membrane seven times (Wikapedia1). Calcitonin is able to bind to at least two signal transduction pathways including the CAMP signal transduction pathway and phospholipase C (PLC) enzyme pathway ( Masi, Brandi 5). The CAMP signal transduction pathway utilizes a G protein called Gs and this G protein consists of a beta, alpha and lambda subunit (Norris, Carr 54). The alpha subunit of the GS G protein will work with the enzyme adenylyl cyclase to produce the second messenger CAMP (Norris, Carr 54). When this G coupled receptor lacks a ligand (in this case calcitonin) GDP will be bound to the GS G protein and the enzyme adenylyl cyclase is inactive (Norris, Carr 54). However, when a ligand binds to the G protein coupled receptor GDP will be exchanged for GTP and the new GTP will bind to the alpha subunit of the G protein (Norris, Carr 54). The alpha subunit can then dissociate from both lambda and beta subunits and interact with adenylyl cyclase to produce CAMP. To turn off this receptor the alpha subunit will convert GTP back to GDP in order to decrease the activity of adenylyl cyclase and therefore the production of CAMP (Norris, Carr 54). The alpha subunit will then recombine with both Lambda and beta subunits to wait for the next ligand to bind to the G protein Coupled receptor (Norris, Carr 5). It is important to note that in this signal transduction pathway CAMP act as a second messenger to amplify and carry the bioregulators signal into the cell. The Phospholipase C signal transduction pathway is completed through the use of a G protein named GQ. During this signal transduction pathway phosphatidylinositol biphosphate (PIP2) is cleaved, leading to the production of Inositol triphosphate (IP3) and diacylglycerol (DAG) (Norris, Carr 55). These molecules will act as second messengers in this pathway. IP3 will cause calcium to be released from the endoplasmic reticulum and this calcium can also act as a second messenger (Masi, Brandi 5). DAG and calcium will turn on a protein kinase C to activate enzymes in the cytoplasm of the cell (Norris, Carr 55). Both calcitonin gene related peptide and calcitonin receptors are a part of the 15 human family B (or Secretin‐like) GPCRs. This subfamily will form complexes with receptor modifying (RAMP 1,2, 3) and “RAMP association with the CTR or with CLR generates multiple distinct receptor phenotypes with different specificities for the CT peptide family” (Barwell 2). Both calcitonin and calcitonin gene related peptide can use the CAMP or phospholipase C signal transduction pathways. RAMP’s interact with calcitonin and calcitonin gene related peptide receptors to change their selectivity to certain ligands and therefore their function in the cell. An association with RAMP one creates the calcitonin gene related peptide receptor while adding RAMP one, two, or three causes the calcitonin receptor to take on the function an Amylin receptor (Barwell et. all 2). Amylin is a 37 amino acid peptide that functions to inhibit insulin secretion, glucose transport to skeletal muscles, gluconeogenesis, and gastric emptying (Masi, Brandi 1). Amylin also contains some capabilities to modulate calcium excretion by decreasing calcium reabsorption (Masi, Brandi 1). The only reason for differentiation between calcitonin and calcitonin gene related peptide is the inclusion of RAMP one into the calcitonin gene related peptides receptor (Barwell et. all 2). The calcitonin receptor does not exhibit a RAMP protein when it is preforming the actions of interfering with regulation calcium and phosphate metabolism. Both receptors contain a large N Terminal Extracellular Domain (ECD), 3 extracellular loops (ECL 1,2,3), 7 transmembrane domains, 3 intracellular loops (ICl 1,2,3) and an intracellular C terminus (Barwell et. all 3). “The concept that most simply encapsulates the mode of ligand binding and activation of family B GPCR is known as the two domain model” (Barwell, et. all 3). In this model the C terminus of a ligand is captured by the ECD, brought to the ECL and upper transmembrane domain of the receptor where the receptor will undergo a conformational change (Barwell, et. all 3). This will allow the receptor to be turned on and second messenger production to carry the bioregulators signal. The three intracellular loops and receptor C terminus are responsible for interacting with the GQ or GS protein to carry out the signal transduction pathway (Barwell, et. all 3). The ECL and the upper transmembrane domain referred as the juxtamembrane region and the ECD is the site where the ligand will bind to its receptor. “The family B GPCR ECDs all share the same overall fold consisting of two antiparallel β‐sheets and an N‐ terminal α‐helix that is stabilized by three disulfide bonds” (Barwell, et. all 3). Peptides that bind to the ECD will adopt a helical conformation and have stabilizing hydrophobic and electrostatic interactions with its receptor (Barwell, et. all 3). A diagram of the calcium gene related peptide receptor can be seen in figure 4 and this receptor structure is very similar to Calcitonin’s receptor. Figure 4 The process of metabolizing calcitonin in the body is due to the existence of peptidase enzymes in the blood. Peptidase enzymes will attack one end of the polypeptide hormone and remove its amino acids one at a time to decrease the biological effects of this hormone (Norris, Carr 59). The entire hormone will eventually be lost through this process and the peptide hormone is no longer able to bind to its receptor (Norris, Carr 59) “CT has a short biological half‐life (five minutes) related to the presence of peptidases in the blood and the fact that all fragments of CT are inactive” (Norris, Carr 509). References Barwell, J., et.all. (2012). Calcitonin and Calcitonin Receptor‐like Receptors: Common Themes with Family B GPCRs. US National Library of Medicine & National Institutes of Health, 51‐65. Carr, J., & Norris, D. (2013). Vertebrate Endocrinology. Walthman, MA: Elsevier Inc. Masi, L., & Brandi, M. (2007). Calcitonin and Calcitonin Receptors. US National Library of Medicine & National Institutes of Health, 117‐122. Pondel, M. (2000). Calcitonin and Calcitonin Receptors: Bone and Beyond. US National Library of Medicine & National Institutes of Health, 405‐422. Szkudinski, M. Thyroid Stimulating Hormone and Thyroid Stimulating Hormone Receptor Structure and Function. Vol. 82 473‐502 Retrieved 19 January 2014 from OLPC: www.wikipedia.org/wiki/Calcitonin Pateu, M. (2008). Calcitonin. www.maptest.rutgers.edu