When a peptide protein based hormone binds to receptors on the surface of a cell?

Abstract

Peptide hormones represent a major class of hormones that are made from amino acids by specialized endocrine glands. The maturation of bioactive hormones take place in the rough endoplasmic reticulum and Golgi apparatus, where preprohormones are proteolytically cleaved into prohormones, and subsequently into mature peptide hormones. Once the bioactive hormones are released into the circulation, they interact with receptors located on the plasma membrane of target cells, and initiate intracellular signaling pathways to regulate physiological processes including energy metabolism, growth, stress, and reproduction. However, excessive amount of circulating peptide hormones often associates with the presence of tumors. Section 2 discusses 10 peptide hormones as tumor markers and their clinical application in aiding the diagnosis of tumors as well as monitoring the disease process.

I INTRODUCTION

Peptide hormones released from the anterior pituitary bind to specific receptors on a limited number of cell types (steroidogenic cells). Signals resulting from this binding are amplified through the production of steroid hormones, leading to the regulation of transcription of genes in all cells. A major advancement in biology has been the identification and characterization of nuclear receptors that bind specific ligands, forming complexes that bind to specific DNA sequences through their zinc finger motifs and thereby regulating transcription of the associated genes. Levels of one class of ligands, the steroid hormones, are controlled by the action of peptide hormones from the anterior pituitary. Over the same period of time that the nuclear steroid hormone receptors have been characterized, an understanding of the regulatory processes leading to production of these steroidal ligands has emerged. Consequently, we now have a good view of how these peptide hormones exert their actions. Adrenocorticotropin (ACTH) receptors are found in the adrenal cortex, luteinizing hormone (LH) receptors in the testis and ovary, and follicle-stimulating hormone (FSH) receptors in the ovary. Each of these endocrine tissues is a factory for production of a specific subset of steroid hormones. In this way the endocrine roles of the adrenals and gonads serve to amplify the action of peptide hormones in a few cells to regulate gene transcription in all cells (Fig. 1).

Each of these peptide hormone receptors is coupled to adenylate cyclase through G-proteins, and consequently binding of the appropriate peptide hormone activates production of cAMP, which in turn activates steroid hormone biosynthesis by acute and chronic mechanisms. Both mechanisms are mediated by cAMP through cAMP-dependent protein kinase (PKA), and the aim of this article is to provide an overview of both actions of peptide hormones in the regulation of steroid hormone biosynthesis.

Concluding Remarks

Peptide hormones exhibit a wide range of posttranslational modifications. Some trends correspond to the protease processing groups. Disulfide bonds and glycosylation of mature hormone peptides are largely found in Group A and B hormones. Combinations of endoprotease cleavage, carboxypeptidase cleavage, and N- and C-terminal modifications are associated largely with Groups C, D and E, largely because the basic cleavage sites remaining at the C-termini require subsequent processing. While most peptide hormone secretion is largely via the regulated secretory pathway, at least one hormone, FSH, is secreted constitutively. Constitutive secretion of other hormones may also complement regulated secretion of other hormones. Similarly, posttranslational translocation from the cytoplasm to the ER lumen provides an alternative to SRP-mediated transfer into the ER lumen for short translation products. Redundancy in peptide hormone processing complicates working out the mechanisms responsible for the synthesis of individual hormones, but provides resilience to cells and individuals.

Peptides

Peptide hormones are polymers of small numbers of amino acids (from fewer than ten to a few hundred); in other words, they are small proteins. Like monoamines, they generally utilize membrane-bound receptors, often GPCRs. The complexity of peptide hormones means that they often show variation in exact structure according to the organism examined. Steroids and monoamines are chemically identical from species to species (e.g., testosterone in a fish is identical in structure to testosterone in a human), although there are species differences in the nature and pattern of synthesis and release of the hormone (e.g., in many male fish 11-ketotestosterone is the dominant circulating androgen and is produced by steroidogenic enzymes not present in humans).

2.1.1 Comparative Genomics

Most peptide hormone genes are single copy and produce mature hormones that are monomeric. However, differential processing can yield multiple bioactive forms for some peptide hormones with the most extreme example in mosquitoes being prepro-FMRFamide, which is processed into nine functional FMRFamide isoforms (Predel et al., 2010). Some peptide hormone genes along with their receptors are duplicated in mosquitoes and select other insects. These include adipokinetic hormone (AKH), corazonin, and AKH/corazonin peptide (ACP) (Table 1). Some of these duplications, however, have been lost in drosophilids including D. melanogaster (Hauser and Grimmelikhuijzen, 2014; Vogel et al., 2013). The only multimember peptide hormone genes in mosquitoes, drosophilids, and other insects are the insulin-like peptides (ILPs), but it is unclear all family members derive from a common ancestral gene (Grönke et al., 2010). Lastly, bursicon and glycoprotein A2/B5 are dimeric peptide hormones that are produced from cleavage products of different prepropeptides (bursicon/partner of bursicon and glyprotein hormone A2/A5), whose corresponding genes are also expressed in different neurosecretory cells (Table 1). Trypsin modulating oostatic factor (TMOF) is a proline rich, 10 amino acid peptide identified in Ae. aegypti that is often discussed as a peptidyl regulator of blood meal digestion and egg formation in mosquitoes (Borovsky et al., 1990; Verlinden et al., 2014). However, TMOF is not included in Table 1 or further discussed here because it matches a 10 amino acid sequence of the vitelline envelope protein (Lin et al., 1993), which clearly is not a peptide hormone precursor protein. In addition, no data compellingly supports that TMOF binds a specific receptor including any GPCR, PRK, or RGC, or activates signalling in any target cell that is consistent with it functioning as a peptide hormone.

Table 1. Peptide Hormone Genes for Three Mosquito Species and D. melanogastera

Overall, the data in Table 1 show Ae. aegypti, An. gambiae, and C. quinquefasciatus encode the same peptide hormone genes with two exceptions. First the short neuropeptide (sNPF) gene in Ae. aegypti has duplicated, which we refer to here as sNPF1 and sNPF2 (see Section 4.1.16), whereas An. gambiae and C. quinquefasciatus have only one sNPF gene. Second, Ae. aegypti has eight ILP genes while An. gambiae has seven and C. quinquefasciatus has five. Table 1 notes a few other instances of genes not being detected in C. quinquefasciatus but this is likely due to annotation issues. D. melanogaster lacks genes for ACP, allatotropin, and OEH but orthologs of all other peptide hormone genes in mosquitoes are present. Orthologs of most peptide hormone genes in mosquitoes and drosophilids are also present in insects in other orders. The proctolin gene is present in D. melanogaster and other insects but is absent in mosquitoes. A few other peptide hormone genes present in one or more insects from other orders are absent from both mosquitoes and drosophilids (Table 1). The latter could indicate these peptide hormones are absent from all of the Diptera.

N Terminus

Peptide hormones are sometimes modified by N-acetylation, as in the case of α-MSH. This modification results from the transfer of an acetyl group from acetyl coenzyme A to the N-terminal amino group. Other N-terminal modifications result from cyclization of N-terminal glutamine to pyroglutamic acid. Such modifications enhance stability by preventing the action of aminopeptidases. In very rare instances, amino mono- or dipeptidases act on precursor proteins following their maturation to remove interfering N-terminal residues. Examples are the activation of the yeast α mating factor and honey bee promellitin by an aminodipeptidase, which removes several successive Glu-Ala dipeptides as secondary cleavages during the maturation of these biologically active peptides.

Abstract

Peptide hormones and growth factors initiate signalling by binding to and activating their cell surface receptors. The activated receptors interact with and modulate the activity of cell surface enzymes and adaptor proteins which entrain a series of reactions leading to metabolic and proliferative signals. Rapid internalization of ligand–receptor complexes into the endosomal system both prolongs and augments events initiated at the cell surface. In addition endocytosis brings activated receptors into contact with a wider range of substrates giving rise to unique signalling events critical for modulating proliferation and apoptosis. Within the endosomal system, receptor function is regulated by lowering vacuolar pH, augmenting ligand proteolysis and promoting receptor kinase dephosphorylation. Ubiquitination–deubiquitination plays a key role in regulating receptor traffic through the endosomal system resulting in either recycling to the cell surface or degradation in multivesicular–lysosomal elements. From a clinical perspective there are several studies showing that manipulating endosomal processes may constitute a new therapeutic strategy.

Discovery of RGF

Secreted peptide hormones in plants often undergo complex posttranslational modifications that are mediated by specific enzymes, which recognize particular sequences of multiple target peptides. Because such modifications are generally critical for the functions of individual peptide hormones, the presence of novel peptide hormones should be revealed through phenotypic analysis of the mutants of posttranslational modification enzymes.

Tyrosine sulfation is a posttranslational modification that has been found in peptide hormones in both animals and plants.9 This modification is mediated by tyrosylprotein sulfotransferase (TPST), which catalyzes the transfer of sulfate to the phenolic group of tyrosine. Arabidopsis TPST (AtTPST) is a type I transmembrane protein localized in cis-Golgi.5 The loss-of-function mutant of AtTPST (tpst-1) shows stunted root phenotype accompanied by loss of maintenance of stem cells and a considerable decrease in meristematic activity (Fig. 1). Because AtTPST is a single copy gene, phenotypes of tpst-1 mutant should reflect the deficiency in the biosynthesis of all the functional tyrosine-sulfated peptides found in Arabidopsis. Interestingly, two sulfated peptide hormones, PSK7 and PSY1,2 promoted cell elongation activity of tpst-1 roots but did not restore the meristematic activity, indicating that tyrosine-sulfated peptide(s) other than PSK and PSY1 is involved in the maintenance of root stem cells and regulation of meristematic activity.

To identify this peptide signal, genes encoding possible sulfated peptides in the Arabidopsis genome were screened by an in silico approach. Known sulfated peptide hormones, PSK and PSY1, are encoded by multiple paralogous genes whose primary translated products are about 70- to 110-amino acid cysteine-poor (Cys < 6) peptides/polypeptides that contain secretion signal and Asp-Tyr sequences as a minimal tyrosine sulfation motif.2,6 Based on these criteria, 34 candidate peptides/polypeptides were selected from 31,835 open reading frames in Arabidopsis. The RGF family was identified by determining their mature peptide structures followed by testing their activities to recover root meristem defects of tpst-1 mutant.8

1 Introduction

Peptide hormones mediate organ-to-organ communication to help maintain metabolic homeostasis. These factors are released from cells of origin and travel via the circulation to distant target sites where they interact with surface membrane receptors to elicit biological responses through signal transduction systems. The class B G-protein coupled receptors (GPCRs) are a major family of receptors that bind peptide hormones to mediate control of many physiological processes, including regulating growth, managing the stress response, and controlling glucose metabolism and energy balance (Hoare, 2005). Similar to other types of GPCRs, these receptors contain seven transmembrane-spanning α-helices connected by intracellular and extracellular loops and a cytoplasmically located C-terminus.

The distinguishing feature of class B receptors is a large N-terminal extracellular ectodomain (ECD) that is at least 100 amino acids in length for all 15 members of the receptor family (Zhao et al., 2016). The ECDs are globular structures forming tri-layer α-β-βα protein folds that are stabilized by three pairs of disulfide bonds formed between six conserved cysteine residues. The disulfide bonds lock the ECD layers into conformations that enable use of these domains as “affinity traps” to recognize and bind cognate peptide ligands for the various receptors. Subsequent interactions between a docked ligand and the helical bundle of the receptor core occur to promote movement of the transmembrane helices that allow interaction with G-protein to induce signaling. Overall, the receptor activation mechanisms for class B GPCRs requires the peptide hormones to make many high-affinity contacts with the receptor over large interaction surfaces (Bortolato et al., 2014).

The glucagon-like peptide-1 receptor (GLP-1R) is one of the preeminent class B GPCRs as both its physiologic control of insulin secretion and its ligand binding-receptor interactions using structural biology have been well-studied, and there are several GLP-1R agonists registered for treating type 2 diabetes mellitus (Jazayeri et al., 2017; Liang et al., 2018; Sloop, Emmerson, Statnick, & Willard, 2018; Song et al., 2017; Zhang et al., 2017). These drugs improve glycemic control, reduce body weight, and some have been shown to provide cardiovascular health benefits (Gerstein et al., 2019; Hernandez et al., 2018; Marso, Bain, et al., 2016; Marso, Daniels, et al., 2016). All of these medicines are peptide-based, therefore requiring subcutaneous injection, but the therapeutic success of GLP-1R agonists justifies investigating additional therapeutic modalities targeting this receptor. These include parenterally administered multi-functional agents (peptide-based GLP-1R agonists that also activate other receptors) and orally bioavailable non-peptide agonists or positive allosteric modulators that may enhance the actions of endogenous GLP-1R ligands (Mendez et al., 2019; Willard, Briere, & Sloop, 2018).

1 Introduction

Peptide hormones have widely been studied in the field of life sciences and used in therapeutic applications. There are currently more than 40 commercially available peptide-based drugs such as insulin, and ANP (atrial natriuretic peptide) and GLP-1 (glucagon-like peptide-1) analogs. In addition, over 100 new peptide therapeutics are currently being evaluated in clinical trials (Reichert, 2010). Their molecular weights differ dramatically from those of small molecules, such as the tripeptide TRH (thyrotropin-releasing hormone) analog, and of long polypeptides such as human insulin (51 residues) and PTH(1–84) (parathyroid hormone). Because of their range in size, many different methods are used to produce these peptides, such as chemical synthesis, recombinant expression, fermentation, and extraction from native tissues. Chemical synthesis or recombinant expression system is commonly utilized for medium- or large-scale preparation.

Most biologically active peptide hormones have a wide variety of posttranslational modifications such as C-terminal amidation, phosphorylation, and acetylation. These modifications are very important for their biological activity and stability in the blood stream (Matsubayashi, 2011; Reichert, 2010). Ghrelin is an acylpeptide consisting of 28 amino acids, with Ser3 esterified with octanoic acid (Kojima et al., 1999). This modification is essential for its biological activity (Kojima et al., 1999; Matsumoto et al., 2001a).

Here, we provide two different protocols for human ghrelin preparations, namely, a chemical synthesis method and a semisynthesis method on a laboratory scale.

For scales up to several hundreds of milligrams, a solid-phase chemical synthesis protocol can be utilized for the preparation of human ghrelin, as shown below. However, preparation at the gram scale is still challenging because (1) chemical synthesis imposes a limitation on the size of peptide that can be produced on a large scale and (2) even with recombinant expression, which is more suitable for the large-scale preparation, the majority of the commonly utilized expression hosts do not have endogenous posttranslational modification machinery for ghrelin.

Therefore, we also provide a semisynthesis method, combining chemical synthesis and recombinant expression system, that utilizes the advantages of both (Makino et al., 2005).