1 Title: "POMC; the physiological power of hormone processing" 2 3 4 5 Erika Harno1, Thanuja Gali Ramamoorthy1, Anthony P. Coll 2 and Anne White1 6 7 8 9 1Division of Diabetes, Endocrinology and Gastrointestinal Sciences, Faculty of 10 Biology, Medicine and Health, University of Manchester, Manchester, UK and 11 2MRC Metabolic Diseases Unit, Wellcome Trust-MRC Institute of Metabolic 12 Science, Cambridge, UK 13 14 15 16 17 18 Correspondence to: 19 Professor A. White, 20 Division of Diabetes, Endocrinology and Gastroenterology, 21 School of Medical Sciences, 22 Faculty of Biology, Medicine and Health, University of Manchester, 23 Manchester Academic Health Sciences Centre, 24 3.016 AV Hill Building, 25 Manchester, 26 M13 9PT, 27 UK. 28 Tel.: +44 (0)161 275 5178 29 E-mail address: [email protected] 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 1 50 TABLE OF CONTENTS 51 52 I. INTRODUCTION 53 II. OVERVIEW OF POMC PROCESSING 54 III. ENZYMES INVOLVED IN PROCESSING POMC TO DIFFERENT PEPTIDES 55 IV. THE CELLULAR PATHWAY TO SECRETION 56 V. RECEPTORS BINDING POMC-DERIVED PEPTIDES 57 VI. ROLES OF THE COMPONENT PEPTIDES OF POMC 58 VII. DISORDERED PROCESSING IN THE HYPOTHALAMUS; CHILDREN WITH 59 OBESITY 60 VIII. DISORDERED PROCESSING IN THE HYPOTHALAMUS; MICE WITH 61 OBESITY 62 IX. POMC PROCESSING IN THE SKIN 63 X. PITUITARY PROCESSING OF POMC: A KEY FACET IN REGULATION OF 64 THE HPA AXIS 65 XI. POMC PROCESSING BY TUMORS 66 XII. ASSAYS FOR POMC DERIVED PEPTIDES – WHAT ARE YOU REALLY 67 MEASURING? 68 XIII. BIOACTIVITY OF POMC AND DERIVED PEPTIDES 69 XIV. CONUNDRUMS AND FUTURE PERSEPECTIVES 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 2 99 ABSTRACT 100 101 Pro-opiomelanocortin (POMC) is the archetypal polypeptide precursor of 102 hormones and neuropeptides. In this review, we examine the variability in the 103 individual peptides produced in different tissues and the impact of the 104 simultaneous presence of their precursors or fragments. We also discuss the 105 problems inherent in accurately measuring which of the precursors and their 106 derived peptides are present in biological samples. We address how not being 107 able to measure all the combinations of precursors and fragments quantitatively 108 has affected our understanding of the pathophysiology associated with POMC 109 processing. To understand how different ratios of peptides arise, we describe the 110 role of the pro-hormone convertases (PCs) and their tissue specificities and 111 consider the cellular processing pathways which enable regulated secretion of 112 different peptides that play crucial roles in integrating a range of vital 113 physiological functions. In the pituitary, correct processing of POMC peptides is 114 essential to maintain the hypothalamic-pituitary-adrenal axis and this processing 115 can be disrupted in POMC expressing tumours. In hypothalamic neurons 116 expressing POMC, abnormalities in processing critically impact on the regulation 117 of appetite, energy homeostasis and body composition. More work is needed to 118 understand whether expression of the POMC gene in a tissue equates to release 119 of bioactive peptides. We suggest that this comprehensive view of POMC 120 processing, with a focus on gaining a better understanding of the combination of 121 peptides produced and their relative bioactivity, is a necessity for all involved in 122 studying this fascinating physiological regulatory phenomenon. 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 3 148 I. INTRODUCTION 149 150 A. The Discovery of POMC as a Precursor 151 The phenomena of POMC as a hormone precursor emerged gradually over time 152 as observations slowly filled in pieces of the puzzle. Long before the concept of 153 hormone precursors was realized, the bronzed skin color described by Addison 154 in his patient with adrenal insufficiency (“melasma suprarenale”) gave perhaps 155 the first hints of a connection between the hypothalamic, pituitary, adrenal 156 (HPA) axis and skin colour. A similar link between the pituitary and 157 pigmentation came from the studies of Allen and Smith (5, 376) who both noted 158 that immersing tadpoles in pituitary extract made their skins darker. In humans 159 too, large doses of porcine pituitary extract also appeared to cause pigmentation 160 (218), with this active extract of the pars intermedia of the pituitary henceforth 161 termed “melanocyte stimulating hormone” or MSH. 162 163 In 1932, Cushing extended his clinical reports of a polyglandular syndrome 164 caused by basophilic adenomas of the pituitary by linking this finding with 165 adrenal hyperactivity. In the 1930’s work by Ingle and Kendall (177) showed 166 that administration of large amounts of “cortin” , a purified adrenal extract, 167 produced atrophy of the adrenal cortex in rats. Importantly they found that 168 administration of the “adrenotropic principle” of the anterior pituitary was 169 effective in preventing adrenal cortical regression following treatment with 170 cortin. The first hints of a behavioural angle to pro-opiomelanocortin (POMC) 171 biology came from studies by Ferrari in the 1950s, when “stretching-yawning 172 syndrome” – a bizarre crisis of muscular tone - occurred following central 173 administration of MSH. Many other studies assessing the effects of central -MSH 174 on motivational processes followed but it was not until 1976 that Panskepp 175 observed for the first time that this peptide decreased food intake (294). 176 177 Viewed from the comfort and assured knowledge of the modern molecular world 178 these observations and interventions could be considered overtly simplistic. 179 However we believe that these classic observations should be regarded as 180 essential building blocks not only for our understanding of POMC peptide 181 processing but also for the work which subsequently tied together these 182 seemingly diverse peptides. 183 184 B. The emergence of the precursor paradigm 185 186 It is likely that POMC arose over 500 million years ago by an insertion of the 187 melanocortin sequences into a prepro-endorphin gene. Evidence for this comes 188 from structural identities with other opioid precursors in both the N- and C- 189 terminal regions of POMC (266). The common opioid gene was thought to arise 190 during chordate evolution. There are four opioid genes which are on three 191 chromosomes in the vertebrate genome. An intragenic duplication event in 192 tetrapods is thought to have led to the presence of -MSH, -MSH and γ-MSH 193 (265). The -MSH sequence is not present in teleosts and is found as a vestige in 194 non-teleosts, whereas an additional melanocortin peptide, termed δ-MSH has 195 been found in cartilaginous fish. This suggests a divergence in MSH sequences in 196 cartilaginous, ray and lobe-finned fish (266). 4 197 198 The golden age for the precursor paradigm came in the 1960s and 1970s 199 particularly when the first evidence for a precursor of insulin was unearthed by 200 Don Steiner and his team (382, 383). Sequencing confirmed the existence of pro- 201 insulin in 1968 (60) and subsequently pro-insulin was shown to be relatively 202 less active compared to insulin (202). This inspiring work by Don Steiner paved 203 the way for a much greater understanding of a whole range of pro-hormones 204 particularly in relation to their processing. 205 206 High molecular weight forms of ACTH and -LPH: Although 207 adrenocorticotropic hormone (ACTH) and -lipotropin (-LPH) had been 208 characterized separately, the concept that they were produced as part of a 209 common precursor had not been considered and only emerged after a number of 210 different approaches suggested the sequences for these different peptides in the 211 same molecule (Figure 1) (reviewed in (66, 280)). Elegant studies by Yalow and 212 Berson (433) using normal human pituitary extracts and an ectopic ACTH 213 producing thymoma, indicated that ACTH was present in a high molecular 214 weight form. These high molecular weight forms of ACTH were also identified in 215 the mouse pituitary tumor cell line, AtT20 (119, 232). Lowry et al. (230) went on 216 to use human pituitary extracts and precipitated a single pro-hormone using 217 antibodies to the different peptides (228). This was made possible because 218 previous work by Chrétien and Li (65) had discovered that the -LPH sequence 219 was found within -LPH and that it had the -MSH sequence at its C-terminal. 220 This led them to propose a pro-hormone theory (reviewed in (66)). The presence 221 of an opioid peptide at the C-terminal of -LPH was a serendipitous finding by 222 Hughes et al. (173) when they identified the met-enkephalin sequence at the N- 223 terminal of -endorphin in the -LPH molecule. This was confirmed by the 224 sequencing of -endorphin (154). 225 226 Figure 1: Processing of POMC in different tissues 227 228 In 1978, the concept that POMC was a pro-hormone for ACTH and -LPH was 229 confirmed in studies with the ACTH-secreting AtT20 cell line. Mains et al. 230 radiolabeled amino acids in the cells and then used immunoprecipitation and 231 SDS gel electrophoresis, enabling them to identify a 31Kd peptide recognized by 232 antibodies to both ACTH and -LPH (234). Roberts and Herbert utilized a similar 233 approach but with cell-free translation and antisera to both peptides and 234 reported similar results (329). 235 236 The emergence of the full structure of POMC: Not long after these studies, the 237 precursor peptide was purified from rat pituitaries (335) and (rather strangely) 238 from camel pituitaries (200).