CHAPTER 3 BIOSYNTHESIS AND SECRETION OF PEPTIDE HORMONES
Principles and Practice of Endocrinology and Metabolism
CHAPTER 3 BIOSYNTHESIS AND SECRETION OF PEPTIDE HORMONES
WILLIAM W. CHIN
Overview of Peptide Hormone Synthesis and Secretion
Messenger RNA Processing
Regulation of Polypeptide Hormone Synthesis
Generation of Diversity
In the endocrine system, hormones are factors produced by groups of cells clustered in specific tissues, commonly known as glands, and released into the general circulation to affect the function of distant target cells. Because hormones are responsible for the control of a complex metabolic milieu, they, along with the hormone-producing and target cells, participate in intricate regulatory networks (see Chap. 4 and Chap. 5).
An important feature is positive regulation of hormone synthesis and secretion. For example, gonadotropin-releasing hormone (GnRH)from the hypothalamus stimulates production and release of the pituitary gonadotropins. Another common theme is regulation by negative feedback, by which a trophic hormone stimulates the production and secretion of a second hormone in a target cell that acts on the original gland to decrease secretion of the trophic hormone. For example, thyroid-stimulating hormone (TSH) is produced and secreted from the thyrotrope in the anterior pituitary gland. It stimulates the thyroid gland to synthesize and secrete thyroid hormones, which act on the thyrotrope to decrease further production and release of TSH. Thus, hormones may regulate the biosynthesis and release of other hormones. Moreover, hormones may control other cellular activities by determining the amount and activity of other proteins. This regulation occurs largely at the gene transcriptional level, although some regulation occurs at post-transcriptional, translational, and posttranslational levels.
In addition to operating at the broader level of the endocrine system, factors secreted from a given cell can influence cellular activities in adjacent or neighboring cells (i.e., paracrine effects) or within itself (i.e., autocrine effects; see Chap. 1). Intracellular communication effected by hormonal factors is critical for the integrated function of an organism. The pivotal and ubiquitous nature of hormones in these tasks makes understanding their synthesis and release essential.
This chapter describes the events that occur in the biosynthesis of polypeptide hormones in hormone-secretory cells, from the gene to the final, bioactive protein hormone, including the intracellular structures involved in this process. Insights about the regulation of the hormone-producing cell derived from studies involving recombinant DNA technology and molecular and cellular biology are highlighted.
OVERVIEW OF PEPTIDE HORMONE SYNTHESIS AND SECRETION
Proteins are important as the backbones of polypeptide hormones and as integral components of enzymes that participate in the biosynthetic pathways of steroid and thyroid hormones as well as enzymes that participate in intracellular synthetic and degradative actions and in energy generation. They are critical membrane, receptor, and cytoskeletal molecules, and an appreciation of the pathways for polypeptide synthesis and their associated cellular structures is important. This section describes the general pathways of polypeptide synthesis, with an emphasis on the informational flow from the gene to the final functional protein and the cell structures involved in each of the steps in this highway of biochemical events.1,2 and 3 The production of a functional protein hormone requires numerous steps, each one involving modifications or processing of precursor molecules.
The eukaryotic cell consists of two major compartments, the nucleus and cytoplasm (Fig. 3-1 and Fig. 3-2), which are delimited by plasma membranes that are topologically contiguous with one another (see Fig. 3-2). The nucleus is surrounded by a nuclear envelope consisting of an outer and inner membrane encompassing a cisternal space.4 The nuclear envelope is perforated by nuclear pore complexes that permit communication of the nucleoplasm with the cytoplasm.
FIGURE 3-1. Electron micrograph of a rat lactotrope. The rat lactotrope in the anterior pituitary gland synthesizes and secretes prolactin. This electron micrograph reveals a portion of a lactotrope, including the two major compartments, nucleus (N) and cytoplasm. The nucleus is delineated by a double membrane, the nuclear envelope. The key organelles in the synthesis and processing of polypeptide hormones include therough endoplasmic reticulum (r), Golgi stack (G), secretory vesicle (v), and secretory granule (sg). Mitochondria (m), the major source of cellular energy, are shown. The secretory granules are characterized by an electron-dense material representing condensed polypeptide hormone. (Courtesy of Gwen V. Childs, University of Texas at Galveston.)
FIGURE 3-2. Diagram of the secretory cell. The secretory cell contains two major compartments: nucleus and cytoplasm. The nucleus is delimited by a nucleoplasmic membrane that is perforated by nuclear pore complexes. The nuclear membrane is contiguous with the endoplasmic reticulum (ER). Transport of polypeptides from the ER to the next organelle, the Golgi stack, is accomplished by way of transport vesicles or transitional elements. Polypeptide hormones exit from the Golgi stack by formation of secretory vesicles and granules. The stored polypeptide hormone in the secretory granule is released in the process of emiocytosis or exocytosis on receipt of the appropriate extracellular stimuli. The shaded areas represent topologically extracellular spaces. The lumen of the ER and Golgi are contiguous with the extracellular space.
The nucleus contains much of the cellular nucleic acid or genetic material in the form of genes, which harbor the information necessary for the initial production of precursor RNAs and hence functional proteins. The cytoplasm contains multiple organelles that are involved in the synthesis of proteins and their processing. These events occur through an assembly-line arrangement, by which the initial protein precursors are altered by changes in their primary structures and by glycosylation and other chemical modifications. These organelles include the endoplasmic reticulum (ER), where initial protein synthesis occurs, and a complex membranous structure known as the Golgi stack, where further protein processing and posttranslational modifications, sorting, and translocation take place.
The secretory cell is differentiated from other cell types by the presence of secretory granules that emerge from the Golgi stack. These granules are specialized, membrane-bound organelles that contain polypeptide hormones in high concentration that may be stored for long periods. Stimulus-secretion coupling allows release of the hormone from the granule on physiologic demand.
The informational flow from the gene to the final protein is shown in Figure 3-3. Each protein produced by a cell is encoded by a gene. In a typical mammalian haploid genome, ~100,000 genes are grouped together into clusters called chromosomes. However, only a subset of these genes is expressed in a given cell. The genes in chromosomes are organized as chromatin with its DNA bound to basic histone and acidic nonhistone proteins. Specifically, the DNA is wrapped nearly twice around an histone-octamer core at regular intervals (i.e., every 140 nucleotides) to yield nucleosomes and a structure resembling beads on a string. These protein-DNA interactions provide the gene with vital secondary and tertiary structures. The DNA in this condensed form probably is transcriptionally inactive. However, modifications in this structure, dictated by developmental or regulated patterns, may determine whether a particular gene may be transcribed at all and, if so, at what rate. The role of chromatin structure in determining the breadth of cell-specific gene transcription remains to be clarified.
FIGURE 3-3. Informational flow from the polypeptide hormone gene to the bioactive secreted hormone. The gene, containing information in the form of DNA, is transcribed into heterogeneous nuclear RNA. This heterogeneous nuclear RNA is rapidly processed to form mature messenger RNA (mRNA). These events occur in the nucleus. The mRNA enters the cytoplasm, where it interacts with the protein synthetic machinery and undergoes translation, by which the protein hormone precursor is synthesized. Then numerous cotranslational and posttranslational processes occur in the rough endoplasmic reticulum and Golgi stack to yield the mature protein hormone. Secretory vesicles bud and emerge from the trans-Golgi to produce the secretory granule. In this state, the polypeptide hormone is stored and released on stimulation by the appropriate extracellular signals, whereupon the hormone enters the extracellular space. The secreted hormone may be acted on further by extracellular processes to yield other active hormone species and may be subjected to peripheral degradation.
The information in a gene appears in the form of a double-stranded polymer of deoxyribonucleotides (DNA). The protein sequence is embedded in triplets of nucleotides in a tandem array dictated by the genetic code. The information present as DNA in a gene must be transferred to another molecule, RNA, which transfers the original information from the nucleus to the cytoplasm. The informational transfer from the gene to RNA is known as transcription. Initially, a precursor RNA, called heterogeneous nuclear RNA (hnRNA), is synthesized using the original DNA in the gene as a template. This precursor molecule is then rapidly processed into the mature messenger RNA (mRNA).
The mRNA exits the nucleus to enter the cytoplasm. The mRNA molecule is a version of the gene with its data represented as a linear sequence of ribonucleic acids. The mRNA quickly interacts with the protein synthetic machinery, the ribosomes of the cytoplasm. The ribosome is a complex structure that contains ribosomal RNAs and its associated proteins. Within this structure, the information present in the mRNA is eventually transferred to protein information. The availability of several adapter molecules known as transfer RNA (tRNA) allows the information in the triplets of nucleotides (i.e., codons) in the mRNA to be converted to amino acids. Because one of four different nucleotides can occupy each position in a triplet codon, 43 or 64 sequence possibilities exist. More codons are available than are necessary to encode the 20 essential amino acids. The genetic code contains redundancy or degeneracy so that a single amino acid may be represented by more than one codon. Because a protein molecule has a beginning and an end, the mRNA must contain information for the start and stop of translation. Important codons include AUG, or initiator methionine, which is the first amino acid of all newly synthesized polypeptides, and UAG, UAA, and UGA, which are termination codons.
Proteins destined for secretion are produced on ribosomes and possess a NH2-terminal signal or leader peptide that interacts with cytoplasmic and ER receptors to mediate rapid ribosome-ER membrane association. This new complex allows newly synthesized proteins destined for secretion to enter the lumen of the ER, which is topologically located outside the cell. Soon after this occurs, cotranslational events take place, and the newly synthesized polypeptide is then sequestered within the cisternal space of the ER. The polypeptide then migrates from the ER through the Golgi stack, where further processing occurs. After the transfer from the cis to the medial to the trans regions of the Golgi complex, the maturing polypeptide hormone is sorted and transferred to the secretory granule. Polypeptides in secretory vesicles and granules are released into the extracellular space by fusion of the vesicle with the plasma membrane and by exocytosis of the contained material. Hormones in secretory granules are stored until the appropriate extracellular signal, generally a calcium flux, is received to prompt the release of the contents of the secretory granule. In response to the chemical signal, the membranes of the secretory granule fuse with the plasma membrane, and emiocytosis (i.e., regulated exocytosis) occurs.
In a series of alterations in precursor RNA and protein molecules, the eventually mature and bioactive polypeptide hormone is synthesized and secreted.
Polypeptide hormones may be encoded by single or multiple genes. Frequently, a hormone requires only intramolecular folding and formation of disulfide linkages in a single protein backbone to form the bioactive molecule. Sometimes, the bioactive hormone is formed by the covalent or noncovalent association of two or more subunits derived from a single gene or multiple genes. An example of the former is insulin, which is initially synthesized as a precursor with polypeptide subunits A and B interrupted by peptide C. However, during its intracellular processing, disulfide linkages are formed between subunits A and B, with the proteolytic cleavage and removal of peptide C. Two subunits are associated in a covalent manner to yield the bioactive insulin molecule. Major examples of the latter case are glycoprotein hormones (i.e., TSH and gonadotropins) (see Chap. 15 and Chap. 16). In this family of hormones, each member consists of two subunits encoded by separate genes located on separate chromosomes. The subunits become associated in a noncovalent manner to form the bioactive dimer.5
Several hormones require proteolytic cleavage of the precursor molecule before the formation of the bioactive product. The major example is adrenocorticotropic hormone (ACTH) and b-lipotropin produced from the precursor preproopiomelanocortin by trypsin-like proteolytic cleavage at dibasic residues. Each polypeptide may require covalent modifications of its polypeptide backbone. In the glycoprotein hormones, each subunit contains several N-linked carbohydrate moieties, and the b subunit of human chorionic gonadotropin contains additional O-linked oligosaccharides. In yet other molecules, the addition of sulfate, phosphate, acetyl, and COOH-amide groups is necessary for full bioactivity. Bioactive peptides found in the brain-gut axis require a COOH-terminal amide group for full activity. This conversion is catalyzed by an a-amidation enzyme on substrate hormones that possess COOH-terminal sequences: X-Y-Gly ®X-Y-NH2.6,7
The gene that encodes the polypeptide hormone is part of a simple or complex transcriptional unit. A simple transcriptional unit (Fig. 3-4) is composed of two major components: structural and regulatory. A simple unit produces a single mRNA, whereas a complex unit may yield multiple mRNAs, some of which may encode different proteins. The structural region encodes information that is ultimately found in mRNA (Fig. 3-5). However, in most eukaryotic genes, the coding region is not contiguous with that in the mRNA. Intervening or extraneous segments of DNA are placed between regions that eventually are found in mature mRNA. The coding regions in genes that are ultimately found in mRNA are known as exons, and the intervening sequences are known as introns. The role and function of introns are unknown, although introns separate functional domains in many polypeptides.8 A similar exon encoding an epidermal growth factor (EGF)–like domain has been detected in genes encoding the low-density lipoprotein receptor, the precursor of EGF, and clotting factors IX and X.9 These and other data suggest that introns may play important roles in the evolution of protein families. Moreover, introns may participate in alternate splicing of exons, leading to increased mRNA and polypeptide diversity.
FIGURE 3-4. The transcriptional unit. Each polypeptide hormone or subunit is encoded by a transcriptional unit. This diagram shows the transcriptional unit that contains structural and regulatory regions. The regulatory region is shown at the 5′-flanking portion of the transcriptional unit. However, such regulatory elements may occur in other parts of the gene, including introns or 3&3039;-flanking regions. The structural region is bounded by the transcriptional initiation or cap site at the 5′ end and the polyadenylation site at the 3′ end. The signals for transcription termination are more than 50 to 200 nucleotides downstream of the polyadenylation site.
FIGURE 3-5. The structural region of the transcriptional unit. The structural region contains DNA information that is completely copied and transcribed into the heterogeneous nuclear RNA or RNA precursor. The important feature of eukaryotic structural regions of the gene is the presence of exons and introns. The exon contains sequences that are retained in the mature messenger RNA; the intron sequences are removed during RNA splicing in the nucleus. The first nucleotide of the structural region is known as the cap site, which is the point at which transcription begins in the first exon. The structural region terminates at the polyadenylation site, which is determined in part by the presence of a polyadenylation signal, AATAAA, located 15 to 20 nucleotides upstream of the polyadenylation site.
The regulatory region contains elements that determine whether a gene is transcribed and, if so, in what quantity (Fig. 3-6). A structural region alone is ineffective in this informational flow. The presence of a regulatory region is obligatory for the expression of its associated gene. The DNA in a gene is not found devoid of associated proteins in nature. Marked secondary, tertiary, and quaternary structure is found in genes located in chromatin, and covalent modifications of nucleotide residues are found within genes. In particular, methylation of cytosine residues may play an essential part in determining whether regulatory regions are “open” for transcription. The regulatory region plays an important role in moment-to-moment regulation of gene expression and in the tissue-specific and developmental programs of gene expression.
FIGURE 3-6. The regulatory region of the transcriptional unit. This diagram shows the important elements of the regulatory region. Only part of the structural region, including its cap site, is shown. The TATA box is located ~25 to 30 nucleotides upstream of the cap site. It is fixed in position and orientation and binds important binding proteins that allow interaction with RNA polymerase II. The upstream promoter element is located 40 to 110 nucleotides upstream of the cap site and binds critical proteins that also interact with the RNA polymerase II. The interplay of the upstream promoter element and TATA box are crucial for basal expression of a given structural region. Another element is the enhancer, which is located a variable distance from the cap site, including locations downstream of the cap site, and is independent of orientation. The enhancer element also binds to DNA-binding proteins that further augment or decrease transcription. Enhancer interactions determine regulation of gene expression above or below basal levels. A special subset of enhancers includes the hormone regulatory element, which mediates the effects of steroid and thyroid hormones and second messengers induced by polypeptide hormones.
The process of transcription, whereby information in the gene as DNA is transformed into information as RNA, produces a large RNA precursor molecule (see Fig. 3-3).10 The initial RNA transcript commences at the 5′ end of the first exon, continues through the other exons and introns in the structural region, and terminates at the 3′ end of the last exon. This hnRNA contains more information than in the mature mRNA because of the inclusion of intron sequences. The enzyme RNA polymerase II performs the transcription reaction using the DNA in the gene as the template. The first nucleotide in the RNA precursor transcript, generally a purine (A or G), is defined as the transcriptional start, also called the cap site. Subsequent ribonucleotides are polymerized during RNA elongation until the transcript is completed in the process of termination.