In mammals PTH is the 84- amino- acid product of a single- copy gene (Figure 1). The gene, which encodes a larger precursor molecule of 115 amino acids, preproparathyroid hormone (preproPTH), is organized into three exons. Exon I encodes the 5’- untranslated region of the messenger RNA, exon II encodes the NH2- terminal pre- or signal peptide and a part of the short propeptide, and exon III encodes the Lys- 2– Arg- 1 of the prohormone cleavage site, the 84 amino acids of the mature hormone, and the 3’- untranslated region of the mRNA (Figure 2). The importance of correct splicing of the primary PTH gene transcript, or premessenger RNA, was emphasized by the identification of a donor splice mutation in the PTH gene in affected members of a family with autosomal recessive isolated hypoparathyroidism, resulting in the loss of exon II which encodes the initiation codon and signal peptide.
Fig1. Amino acid sequence of mammalian PTH. The backbone sequence is that of the human with substitutions in the rat hormone shown at specific sites. Biological activity is a property of the amino- terminal one- third of the molecule [PTH(1- 34)]. The solid circles show those amino acids that are identical in the human and rat PTH and PTH- related peptide (PTHrP) molecules.
Fig2. Comparison of structural organization of the human PTH, PTHrP, and TIP39 genes. Exons are boxed: from left to right, dark grey boxes denote 5’UTRs, white boxes denote presequences, black boxes denote prosequences, light grey boxes denote mature polypeptide sequences, and dark grey boxes denote 3’UTRs.
A second member of the PTH gene family encodes the parathyroid hormone- related protein (PTHrP) which is the responsible causal factor in the majority of cases of hypercalcaemia associated with malignancies. PTHrP plays a critical role in fetal development, especially skeletogenesis, but is not involved in normal calcium homeostatic control in the adult. In postnatal life, PTHrP regulates the epithelial mesenchymal interactions that are critical for development of the mammary gland, skin, and hair follicle. The PTH gene and PTHLH gene that encodes PTHrP map to chromosomes 11p15 and 12p12.1- 11.2, respectively. These two human chromosomes are thought to have arisen by an ancient du plication of a single chromosome, and their respective gene clusters have been maintained as syntenic groups across the genomes of several species. Because of the similarity in NH2- terminal sequence of their mature peptides, their gene organization, and chromosomal locations, it is likely that the PTH and PTHLH genes evolved from a common ancestral gene, with that for PTHrP being the more ancient gene.
The gene for tuberoinfundibular peptide of 39 residues (TIP39), a more distantly related member of the PTH gene family, resides on chromosome 19q13.33. TIP39 is a neuropeptide [13]. The TIP39 gene shares organizational features with the PTH and PTHrP genes having one exon encoding the 5’UTR, one encoding the precursor leader sequence, and one encoding the prohormone cleavage site and the mature peptide (Figure 2).
Transcription of the PTH gene occurs almost exclusively in the endocrine cells of the parathyroid gland, and is subject to strong repressor activity in all other cells. Ectopic PTH synthesis (i.e. syn thesis outside parathyroid tissue) has been documented in only a very few cases of malignancies associated with hypercalcaemia. Activation of genes in a particular tissue is often related to demethylation of cytosine residues, and the PTH gene in para thyroid cells is hypomethylated at CpG residues relative to other tissues. In one of the few cases of true ectopic PTH production, involving a pancreatic tumour, the upstream regions of the PTH gene were abnormally hypomethylated [14]. The human PTH gene has two functional TATA box- controlled transcription start sites, a cyclic AMP response element (CRE), and a negative vitamin D response element (VDRE) in its proximal promoter. While PTH gene transcription is negatively regulated by the hormonally active metabolite of vitamin D, 1,25(OH)2D, any regulation by extracellular calcium remains to be established. Also located distally are sequences that function to silence transcription in non- parathyroid cells. In a further case of ectopic PTH production, an ovarian carcinoma, this repressor regulatory region was replaced by a foreign sequence that allowed inappropriate transcription of the PTH gene to take place.
The human PTH produced by patients with hyperparathyroidism is structurally normal. In a small number of parathyroid tumours examined, the PTH gene sequence is rearranged, and the 5’ flanking region of the PTH gene is placed upstream of the cyclin D1 (CCND1) gene located on the long arm of chromosome 11. This is thought to lead to deregulated expression of the CCND1 gene that contributes to tumour development. However, this type of gene arrangement occurs very infrequently in parathyroid tumours.
A more common event involves the loss or inactivation of the multiple endocrine neoplasia type 1 (MEN1) gene, also on the long arm of chromosome 11. The protein encoded by the MEN1 gene called menin, is a 610- amino acid nuclear protein. Germline mutations in the MEN1 gene cause familial and sporadic MEN1 and are found in 20% of non- MEN1 parathyroid adenomas. Loss of heterozygosity at 11q13 is found in MEN1 tumours and sporadic parathyroid adenomas consistent with MEN1 being a tumour suppressor gene.
A target of the Wnt pathway, β- catenin, encoded by the CTNNB1 gene, is a candidate for involvement in parathyroid neoplasia. Very few of the parathyroid adenomas examined so far have stabilizing missense CTNNB1 mutations suggesting that mutation of the β- catenin gene itself is unlikely to be involved in the initiation or early progression of parathyroid adenomatosis. However, other com ponents of the Wnt signalling pathway (e.g. a constitutively active LRP5 receptor derived from an alternatively spliced mRNA may be implicated in parathyroid tumorigenesis).
Early onset recurrent parathyroid tumours occur as part of the uncommon autosomal dominant hyperparathyroidism and jaw tumour (HPT- JT) syndrome in which parathyroid carcinoma is frequent. The responsible gene, HRPT2 (also known as CDC73), at 1q31.2, encodes a novel transcription factor, parafibromin, of 531 amino acids. Sporadic parathyroid carcinomas very commonly contain somatic mutations of the HRPT2 gene and some of these patients harbour germline mutations. In these cases, genetic testing in family members provides for early diagnosis. Loss of hetero zygosity at chromosome 1q occurs in carcinomas of the familial and sporadic disorder usually by intragenic mutations.
PTH follows a pattern of biosynthesis and of vectorial transport through organelles of the cell similar to that of many other pep tide hormones. It is biosynthesized on the polyribosomes of the rough endoplasmic reticulum (ER) of the parathyroid endocrine cell. The PTH gene encodes a precursor, preproPTH, extended at the aminoterminus of PTH 1- 84 by 31 residues. The NH2- terminal 25- residue portion, characterized by its hydrophobicity, is called the signal, leader, or presequence, and it facilitates entry of the nascent hormone into the cisternae of the ER. One patient with autosomal dominant hypoparathyroidism was reported to have a mutation within the protein coding region of the PTH gene in which there was a single base substitution (T- >C) in exon II, resulting in the re placement of arginine (CGT) for cysteine (TGT) in the signal pep tide. This places a charged amino acid in the hydrophobic core of the signal peptide, leading to inefficient processing of the mutant preproPTH to PTH. In cases like this it is suggested that the mu tant polypeptide acts in a dominant- negative fashion by promoting ER stress leading to apoptosis and pharmacological chaperones may be beneficial in restoring proper processing of the PTH.
Normally, as the signal sequence of the synthesized hormone emerges from the ribosome, it binds to a signal recognition particle that stops further synthesis of the nascent protein. The signal recognition particle carrying the ribosome then binds to an integral membrane protein of the ER, called the docking protein or signal recognition particle receptor. This protein releases the block in protein synthesis, and the nascent peptide is transported across the membrane into the cisternae of the ER. The signal sequence is sim ultaneously removed at the inner surface of the ER, at a glycyl- lysyl bond, by a signalase enzyme. (Therefore, note that under normal circumstances the preproPTH molecule never exists as a complete entity.) The resultant precursor molecule, proparathyroid hormone (proPTH), is extended at the NH2- terminus of PTH 1- 84 by only six amino acids. The pro sequence is necessary for efficient trans location and cleavage of the signal peptide. Once formed, proPTH is transported to the Golgi apparatus.
The prohormone hexapeptide has several basic residues that serve as a recognition sequence to yield the mature hormone. Unlike many other prohormones, proPTH does not contain another sequence at the COOH- terminus and has not been detected within the circulation even in states of parathyroid gland hyperfunction. ProPTH has little bio logical activity until cleaved to create the hormonal form. The con version of proPTH to PTH takes place within the trans- Golgi network rather than the secretory granules as occurs with other prohormones like proinsulin. The enzymes involved include furin and PC7, mammalian proprotein convertases that are related to bacterial subtilisins. Little proPTH is stored within the gland.
The resultant mature 84- amino acid form of the hormone is packaged in secretory granules and transported to the region of the plasma membrane. The hormone is released by exocytosis in response to the principal stimulus to secretion, hypocalcaemia. The calcium ion does not influence the enzymatic cleavages involved in the processing of preproPTH or proPTH.
