60 YEARS of POMC: Biosynthesis, Trafficking, and Secretion of Pro

Home , Lipotropin, Natriuretic peptide, Neuropeptide

56:4

n x cawley and others Biosynthesis of POMC-derived 56:4 T77–T97 Thematic Review peptides

60 YEARS OF POMC Biosynthesis, trafficking, and secretion of pro-opiomelanocortin-derived peptides

Correspondence should be addressed Niamh X Cawley, Zhaojin Li and Y Peng Loh to Y Peng Loh Section on Cellular Neurobiology, Eunice Kennedy Shriver National Institute of Child Health and Human Email Development, National Institutes of Health, Bethesda, Maryland, USA [email protected]

Abstract

Pro-opiomelanocortin (POMC) is a prohormone that encodes multiple smaller peptide Key Words hormones within its structure. These peptide hormones can be generated by cleavage ff hypothalamus- of POMC at basic residue cleavage sites by prohormone-converting enzymes in the pituitary-adrenal axis regulated secretory pathway (RSP) of POMC-synthesizing endocrine cells and neurons. ff POMC sorting The peptides are stored inside the cells in dense-core secretory granules until released ff POMC-derived peptide secretion in a stimulus-dependent manner. The complexity of the regulation of the biosynthesis, ff vesicle trafficking trafficking, and secretion of POMC and its peptides reflects an impressive level of ff prohormone control over many factors involved in the ultimate role of POMC-expressing cells, that processing enzymes is, to produce a range of different biologically active peptide hormones ready for action when signaled by the body. From the discovery of POMC as the precursor to adrenocorticotropic hormone (ACTH) and β-lipotropin in the late 1970s to our current Journal of Molecular Endocrinology knowledge, the understanding of POMC physiology remains a monumental body of work that has provided insight into many aspects of molecular endocrinology. In this article, we describe the intracellular trafficking of POMC in endocrine cells, its sorting into dense-core secretory granules and transport of these granules to the RSP. Additionally, we review the enzymes involved in the maturation of POMC to its various peptides and the mechanisms involved in the differential processing of POMC in different cell types. Finally, we highlight studies pertaining to the regulation of ACTH secretion in the Journal of Molecular anterior and intermediate pituitary and POMC neurons of the hypothalamus. Endocrinology (2016) 56, T77–T97

Introduction

The birth of pro-opiomelanocortin (POMC) stemmed from such as α-melanocyte-stimulating hormone (MSH) are the landmark work of Dr Choh Hao Li at the Hormone derived from ACTH, and β-MSH and β-endorphin from Research Laboratory at the University of California at β-LPH. Based on common amino acid sequences among Berkeley, where he first elucidated the chemistry of these peptides such as α-MSH and β-MSH in ACTH and adrenocorticotropic hormone (ACTH) and subsequently β-LPH, respectively, the hypothesis emerged that ACTH and β-lipotropin (LPH). Thereafter, an accumulation of peptide β-LPH could be derived from a larger precursor consisting sequence studies from many laboratories led to the of both ACTH and β-LPH (for an historical perspective, recognition that a number of biologically active peptides see Lowry 2015). Subsequently, several groups, including

http://jme.endocrinology-journals.org © 2016 Society for Endocrinology This paper is part of a thematic review section on 60 Years of POMC. DOI: 10.1530/JME-15-0323 Published by Bioscientifica Ltd. The Guest Editors for this section were Adrian Clark and Philip Lowry. Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 06:18:27PM via free access

10.1530/JME-15-0323 Journal of Molecular Endocrinology intermediate peptide;RSP, regulatedsecretorypathway. Lollipopsymbols representglycosylationsites. cell- andatime-dependentmanner. ACTH,adrenocorticotropin; MSH,melanocyte-stimulatinghormone;JP, joiningpeptide;CLIP, corticotropin-like Schematic diagramofthebovinePOMC protein.Theprohormoneencodesmultiplepeptidesthatcanbecleaved byprohormoneconvertasesina Figure 1 granulesasthey aretrafficked into dense-coresecretory nascent vesiclesbuddingfromtheTGNthatwillmature the movement oftheproteinthroughERandGolgito pathway(RSP).Therouteinvolves the regulatedsecretory granulesof end upultimatelyinlargedense-coresecretory folding intheER,POMCistransportedthroughcellto synthesis attheroughendoplasmicreticulum(ER)and (α-MSH), andopiate-likeactivityβ-endorphin).After N-POMC(1–48), steroidogenic activity (ACTH), satiety physiological functionssuchasmitogenicactivity dependent manner. Thesepeptidesexhibitdifferent N-POMC(1–48); andβ-LPH,inatissue-cell- that canincludeACTH;β-endorphin; is posttranslationallycleavedintopeptidehormones neurons inthearcuate nucleus ofthehypothalamus.It (ILs) of the pituitary, respectively, as well as in peptidergic melanotrophs oftheanterior(AL)andintermediatelobes POMC issynthesizedinthecorticotrophsand Intracellular trafficking of POMC maturation Intracellular organization ofPOMC Michel Chretien(Crine ACTH–β opiomelanocortin’ wascoinedbyMichelChretienforthe β the existenceofcommonprecursorforACTHand At aboutthesametime,cDNAcloningstudiesconfirmed biosynthesis ofthedifferentbiologicallyactivepeptides. that thesequentialprocessingofthisprecursorledto β pulse–chase experimentsprovidedevidencethatACTHand (Mains &Eipper1979)andours(Loh),employing -LPH (Nakanishi -LPH werederivedfromalargercommonprecursor, and DOI: 10.1530/JME-15-0323 http://jme.endocrinology-journals.org Thematic Review trans-Golgi network (TGN), where it is sorted into -LPH precursor(Fig. 1)Chretien&Mbikay2016). N-POMC(1-26) RSP sortingsignal N-POMC(1–49) 16k N-POMC(1–77) t al.1979).Hence,thename‘pro- et t al.1979), et 23k Biosyntheticintermediate n

γ cawley 3 -MSH Mains and Eipper Mains andEipper α-, andothers Printed inGreatBritain β-, andγ-MSH; Proopiomelanocortin (POMC) JP α -MSH Kelly 1987).Indeed,atthattime,transfectionofVSVG and secretiontriggeredbyexternalstimuli(Burgess& the otherrequiringactivesortingintostoragegranules driven by bulk flow and tied to protein translation and pathwaysweredistinctinthesecells,onebeing secretory (Gumbiner &Kelly1982),demonstratingthatthetwo endogeneous murineleukemiaviruspresentinthesecells cells, thatis,inanunstimulatedmanner, alongwithan pathway(CSP)inthese through theconstitutivesecretory was subsequentlyfoundthatPOMCalsosecreted that POMCmustbeprocessedinthiscompartment.It on Ficoll(Gumbiner&Kelly1981),leadingtotheidea AtT20 cellspurifiedbydensitygradientcentrifugation and evidence demonstratedthatthematurepeptides,ACTH 16-kDa N-POMCintermediate.Initialbiochemical POMC andprocessesitintoACTH,β-LPH,the a mousecorticotrophcelllinethatnormallyexpresses Ideal forstudyingthesequestionsweretheAtT20cells, cellular transportandprocessingoftheprohormone. in the1980sand 1990s addressingthe question of 1977, ACTH andβ-LPH(Mains&Eipper1976, has beenstudiedbymanyinvestigators. granules hasbeenalong-standingquestionandonethat cell fromthesiteofsynthesistodense-core secretory is transportedthroughandprocessedintheendocrine the celluponstimulationbyasecretagogue.HowPOMC core andarestoredinthesegranulesuntilsecretedfrom generated inthematuregranulesformanelectron-dense complement, specificforthatcelltype.Thepeptides convertases (PCs)togeneratethepeptidehormone in atime-andcompartment-specificwaybyprohormone this movementwithinthecell,prohormoneiscleaved to thereleasesitescloseplasmamembrane.During peptides Biosynthesis ofPOMC-derived ACTH Published byBioscientifica Ltd. With ofPOMCastheprecursorto thediscovery CLIP -LPH, were present in mature secretory granules of β-LPH, werepresentinmature secretory Crine t al.1978),anexplosionofworkfollowed et γ -Lipotropin Downloaded fromBioscientifica.com at09/29/202106:18:27PM β -Lipotropin β -MSH β 56 -Endorphin : 4 Mains T78 t al. et via freeaccess Journal of Molecular Endocrinology receptor (Sly&Fischer1982 ). Supportforthisideacame lysosomal enzymesthatuse themannose-6-phosphate event wasbelievedatthattime tobesimilarthatofthe transport through the CSP (Kelly 1985 ). This sorting into theRSPwasdominant over theprocessofbulkflow 1986), demonstratingthatthesortingprocessfor the granulesofRSPinthesecells(Moore&Kelly hGH could directthe constitutively secreted VSVG into of hGHdemonstratedthattheactivesortingprocess of using afusionproteinofVSVGcoupledtotheC-terminus More significantly, animportantgain-of-function study common signalrecognizedbytheAtT20cellmachinery. that otherprohormonesandexocrineproteinscontain a were alsotargetedtotheRSPinAtT20cells,suggesting (Moore exogeneously expressedproteins,includingproinsulin 1984). However, similartothehGH(Moore&Kelly1985), the processingandsecretionofACTH(Burgess&Kelly levels oftheproteoglycans and foundnodifference in of chrondroitinsulfatesynthesiswereusedtoreducethe in thesortingprocesswasdiscountedwheninhibitors these proteoglycansenteringtheRSPcouldparticipate destined tobedirectedintotheRSP. Theideathatmaybe the conclusionthatacommonsignalexistedforproteins those thatfollowedACTH(Moore pathways, those that followed unprocessed POMC and inthetwodistinct AtT20 cellsthatcouldbeobserved and sulfatedproteoglycansidentifiedseveralproteinsin newly synthesizedproteinslabeledwith consistent withthevirusbeinginCSP. Analysisof (Toozewith ACTHbutdivergedafterward murine hepatitisvirussharedthisinitialcompartment al.1987a).Follow-onEMstudiesidentifiedthatthe et immature granules where most of it was processed (Tooze antibody, demonstrating that POMC was sorted into these POMC, using an ACTH-β-LPHcleavage site-specific dense-core granules were shown to contain unprocessed These condensingvacuolesand25–30%ofthemature granule cargowassortedintothese‘immature’granules. apparatus (Tooze &Tooze 1986),demonstratingthat vacuoles protrudingfromthetransmostsideofGolgi showed ACTH-immunoreactivity(IR)incondensing antibody thatcouldlabelACTHanditsprecursor, POMC, and immunocytochemicalanalysesusingananti-ACTH the RSP. Insupportofthis,electronmicroscopic(EM) constitutively, whereasthehGHwassecretedthrough stomatitis virusglycoprotein(VSVG)wassecreted these cells(Moore&Kelly1985),becausethevesicular pathwaysin clearly demonstratedthetwosecretory or humangrowthhormone(hGH)intoAtT20cells http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0323 Thematic Review et al ( Burgess . 1983c)andtrypsinogen n

cawley 35 andothers S inmethionine Printed inGreatBritain t al.1987b), et et al. 1985), (Asp 1986), identifieda3Dmotif(Fig. 2) containingtwoacidic bond pairsinthisdomain(Bennett1984, analyses of N-POMC(1–26) that solved the di-sulfide of N-POMC(1–26),madepossiblebyearlierstructural and Neuro2acells,respectively. Molecular modeling forsortingPOMCtotheRSP inAtT20cells and necessary N-POMC(1–26), containedinformationthatwassufficient demonstrated that the N-terminus of POMC, specifically mutation (lossoffunction)experiments(Cool (Tam on POMC,limitingdomaintransfer(gainoffunction) with areceptor(Loh&Tam 1985).Infurtherstudies (NIL) ofthepituitary, suggestingthatitwasinteracting granulesderivedfrommouseorfrogneuro-IL secretory tightly tomembranesfromenrichedpreparationsof POMC isahighlychargedprotein,itwasfoundtobind solubleinaqueoussolution.Although protein andvery aspartate aminoacids.Hence,POMCisahighlycharged protein iscomposedofarginine/lysineandglutamate/ the POMCprotein.Thus,almostone-thirdof negatively charged(glutamateandaspartate)residuesin residues appear to be physiologically balanced by 39 PC cleavagesites.Interestingly, thesepositivelycharged sequence (withoutthesignalpeptide),manyofwhichare charged (arginineandlysine)residuesthroughoutthe POMC isa31-kDaproteinthatcontains38positively Sorting ofPOMCtodense-core secretory granules importantcomponentofthesortingprocess. as avery used (Dyken&Sambanis1994),supportingtheroleofpH same pHneutralizingeffectonacidiccompartments,were was madelater, whenammoniumions,whichhavethe secretion indicativeofmis-sorting.Asimilarobservation withanincreaseinconstitutive granules wasobserved production ofnewlysynthesizedACTHinthemature compartments. Inthepresenceofchloroquine,reduced (Moore to theRSPwaspreventedinpresenceofchloroquine and recyclingoflysosomalenzymes,sortingPOMC that,similartoapH-dependentsorting from observations and Leu prohormone sortingreceptor. Loh 1998, factor (Lou growth brain-derived neurotrophic factor butnotnerve and hexamericproinsulin (Dhanvantari motif. This motif was subsequently found in monomeric et al. 1995)andpredictedtobeaconsensussortingsignal peptides Biosynthesis ofPOMC-derived Published byBioscientifica Ltd. 10 andGlu t al.1993, et . 1983a),compoundthatneutralizesacidic et al 18 ) residues that were highly conserved (Cool ) residuesthatwerehighlyconserved Loh . 2005),andproenkephalin(Normant& et al 14 t al. 2002), and predicted to bind to a et ) andtwoaliphatichydrophobic(Leu Cool &Loh1994)anddeletionor Downloaded fromBioscientifica.com at09/29/202106:18:27PM 56 : 4 Bennett t al.2003), et et al. 1995) T79 . et al via freeaccess 11

Journal of Molecular Endocrinology acid (GEMSA),apotentinhibitor ofCPE,didnotprevent 1999). Also,additionofguanidinoethylmercaptosuccinic the ligandtosameextent asWTCPE(Zhang active siteofCPE,asCPEwith itsactivesitemutatedbound binding ofN-POMC(1–26) to CPEdidnotrequirethe kinetics (K and characterizeditwithlow-affinityfirst-orderbinding cross-linking andbindingstudiesconfirmeditsidentity (Fricker &Snyder1982, ‘Exopeptidases inPOMCpeptideprocessing’section C-termini ofpeptidehormoneintermediates(seethe removing lysineandarginineaminoacidsfromthe involved inthematurationofpeptidehormonesby was classicallyknownsincetheearly1980sasanenzyme in the eluate identified by amino acid sequencing. CPE (CPE) (Cool receptor forPOMCwasidentifiedascarboxypeptidaseE eluted at pH 7.4. Using this approach, a candidate sorting acidic conditions(pH5.5)andboundproteinswere Solubilized membraneswereappliedtothecolumnunder a putativesource ofa prohormonesortingreceptor. chromatography usingNILGolgi-derivedmembranes, signal motif of POMC, to beads and used it in affinity the N-POMC(1–26) peptide, containing the sorting sorting signal.Notethetwoacidicresidues,Asp An NMRconfomeroftheN-POMC(1–26)peptideencodingRSP Figure 2 two hydrophobicresidues,Leu signal motif. DOI: 10.1530/JME-15-0323 http://jme.endocrinology-journals.org Thematic Review In follow-upstudies,Lohandcolleaguescoupled D ). Inaddition, &Loh1998 (Cool =6µM) . 1997)becauseitwasthemajorprotein et al 11 Hook andLeu . 1982a)).Subsequent et al 18 n , comprisingthesorting

cawley 10 andGlu andothers Printed inGreatBritain 14 , andthe t al. et in Arg of CPEidentifieda putativesortingsignalbinding site enzymatic activityofCPE.Indeed,molecularmodeling POMC toCPEdidnotdependonthecarboxypeptidase binding (Loh WT mice(Cawley almost eight-foldhigherintheCPEKOmicecomparedto resulting inserumlevelsofPOMC/23-kDaintermediate and its23-kDabiosyntheticintermediateintheNIL there isan~10-foldincreaseinthetissuelevelsofPOMC isreducedby81–94%,respectively,of thepituitary and POMC processingtoα-MSHintheNILandhypothalamus (Cawley comes from the analysis of the CPE knockout (KO) mouse indicative of the lack of a sorting receptor. This support 1999) ofthesemicesuggesteddefectivesortingPOMC, AL pituitary primary cells(Cool primary AL pituitary as aCPE-deficientmouse. SecretionstudiesfromNILand Cpe POMCcamefromstudiesonthe a sortingreceptorfor was independentoftheactivesite.SupportforCPEas further demonstratingthatthebindingofN-POMC(1–26) binding sitecomposedofArg indicate theuniqueareastoCPE.Note theN-POMC(1–26)sortingsignal areas indicatethesimilarityofCPEand CPD,whereastheblueareas Molecular modelofCPEbasedonthe crystalstructureofCPD.Thered Figure 3 Cawley unstable andsubjecttodegradation(Varlamov CPE containsaSer202Promutationrenderingtheprotein peptides Biosynthesis ofPOMC-derived Published byBioscientifica Ltd. fat/fat 255 mouse(Naggert et al. 2003).Hence,theCpe and Lys t al.2010).InthecompleteabsenceofCPE, et . 1997),demonstratingthatbindingof et al 260 . 2010).Thissuggestsatrafficking et al of CPE (Zhang 255 Downloaded fromBioscientifica.com at09/29/202106:18:27PM andLys t al.1995)wherethemutant et 260 . t al.1997, et fat/fat i. 3), al. 1999) (Fig. et 56 mousewasviewed : 4 Shen et al. 1997, T80 t al. et via freeaccess Journal of Molecular Endocrinology upregulated in the pituitary oftheseCpe upregulated inthepituitary receptor forchromograninA(Hosaka a CRH-dependentmanner, andSgIII,aknownsorting fromthesemicein released fromtheALofpituitary secretogranin III(SgIII),waspossible,becauseACTH that compensationbyanotherpotentialsortingreceptor, of the authors). Later studies in the but itsprocessingtoACTHwasreduced(unpublisheddata demonstrating thatsomePOMCwassortedintotheRSP significant increasein thestimulated secretion of POMC, ACTH productionandsecretionaswellasmallbut culturesofALcellsfoundsignificantlyreduced on primary Pulse–chaseexperiments content ofPOMCwasobserved. contrast to the NIL, only a small increase in the tissue of POMC to ACTH is significantly reduced; however, in to the phenotype. In the AL of the pituitary, processing defect andaccumulationofthePOMClikelycontributes increased constitutivesecretion,althoughaprocessing defect ofPOMCandaccumulationintheTGNwith receptor for all the prohormones in the cell was that the directing ittothelysosomesfordegradationinvivo. and accumulationofPOMCintheabsenceCPE,by corticotrophs maycompensateforthepoortrafficking authors), indicativeofdegradation.Thissuggeststhatthe 4,unpublisheddataofthe compared toWTcells(Fig. was unaccounted for in the cells from the CPE KO mouse also showed that ~42% of the newly synthesized POMC al.2005).Interestingly, ourpulse–chaseexperiments et of degradation(unpublisheddata theauthors). ACTH-IR waslessintheCPEKOcells comparedtotheWTcellsindicative levels ofACTHmadeintheCPEKO cells. Theoverallrecoveryoftotal analyzed byimmunoprecipitationand quantified.Notethereduced for 30 minandchased2h.Immunoreactive ACTHmoleculeswere and CPEKOmicewerecultured metabolicallylabeledwith Pulse–chase studiesofPOMCinmouseALcells.PituitarycellsfromWT Figure 4 http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0323

ACTH-IR (% total made at T=0) Thematic Review 160.0 100.0 120.0 140.0 20.0 40.0 60.0 80.0 A majorconstraintintheideaofoneproteinbeinga 0.0 POMC2 CPE KO WT

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cawley fat/fat fat/fat t al.2002),was et andothers Printed inGreatBritain mice suggested mice(Hosaka Total 35 S-Met 42% mature secretory granule.Hence,sortingofPOMCto mature secretory hormone readyforsecretion uponstimulationofthe cell canbecarriedout,that is,toprovidethepeptide the ultimateimportantcellular processoftheendocrine and SgIII (Hosaka that canpossiblyinteractwitheachother, suchasCPE mechanism involvingseveralmembrane-boundproteins via theRSP(Cawley secretion of POMC and a reduction in ACTH secretion 2004), also causedasignificant increasein constitutive sorting receptorforchromograninA(Hosaka to bindPOMC(Hosaka 2015). Notably, knockdownofSgIII,previously shown Behrend 2010)andintheother,. itwasnot(Cawleyet al one case,ACTHsecretionwasnormal(Kemppainen& indicative of inefficientsorting totheRSP;however, in was significantlyelevatedwhenCPEknockeddown secretion. Inbothcases, POMC secretionthroughtheCSP conflicting resultsinAtT20cellswithrespecttoACTH t al.1991, et reduced pH,conditionsexpectedintheTGN(Chandra is enhancedinthepresenceofincreasedcalciumand and multimers(Cawley and heterotypicallywithproenkephalintoformdimers suggested that POMC can loosely aggregate homotypically granules.Insupportofthis,evidence maturing secretory peptide hormoneaggregationandcondensationinthe early inthesortingprocesswouldbedistinguishedfrom followed bybindingtoareceptor. Thisphenomenon of prohormonesmayallowconcentrationthecargo that homotypicandevenheterotypicoligomerization didnotfavorit.Itwasthereforeproposed stoichiometry Huttner 1991, these mildacidicandhigh-calciumconditions(Chanat& shown thatmanyRSPcargoproteinsaggregateunder from constitutivelysecretedproteins,becauseithasbeen to answerhowprohormonesaresortedattheTGNaway into thegranulesofRSPisonehypothesisproposed into thegranulesofRSP. Aggregation-inducedsorting cargo withthemembraneformofCPEtoinitiatesorting thus allowingittoactasabridgeconnect the aggregated N-POMC(1–26) domainwasnotrequiredforaggregation, Cpe knocked downcells,supportingthefindingsfrom wasseeninthe for ACTH-IRbyimmunocytochemistry was secreted constitutively and no punctate staining siRNA toreduceCPEinNeuro2acells,transfectedPOMC specifically reducetheexpressionofatargetgene.Using including CPE(Rindler1998). peptides Biosynthesis ofPOMC-derived Published byBioscientifica Ltd. fat/fat RNA interference technology is a powerful tool to mouse(Normant&Loh1998).Laterstudiesshow Seksek Colomer t al.2005),or interchange, so that et t al.1995).Moreimportantly, the et . 2015 ). Theseresultssuggesta et al . 2000),andthisaggregation et al . 2005)andalsoknownasa et al Downloaded fromBioscientifica.com at09/29/202106:18:27PM t al. 1996, et 56 e al.2002), Jain et : 4 . 2002, et al T81 via freeaccess

Journal of Molecular Endocrinology pulldown andcoprecipitation experiments,whichshowed Confirmation ofthisinteraction camefrombiochemical of CPEinteractedwithdynactin (unpublisheddata). yeast two-hybridstudiesshowed thattheC-terminus al.2003).Subsequent endocytosis (Arnaoutova et small cytosolicGTPaseinvolvedinclathrin-independent traverse thegranulemembraneandinteractwithArf6, a identified thattheC-terminusofsomeCPEcould detail untilrecently. PreviousworkbyLohand colleagues vesicles alongmicrotubuleshasnotbeenstudied in al . 1989).Transport ofPOMC on microtubules(Kreiset movement, reportedatarateof3–5µm/s,wasdependent direction and some in the retrograde direction, and the the ACTH-containingvesiclesmostlyinanterograde movement of microscopy demonstrated the saltatory studies usingacridineorangeandenhancedfluorescence on themicrotubules(Tooze &Burke1987).Other during cytokinesistelophase,aprocessdependent anaphase butthenalignatthemidbodyasitdevelops interphase, randomly distributed during metaphase and being localizedattheGolgiandtipsofprocessesin granules containing ACTH redistribute in the cell, from on thatduringcelldivision,thematuredense-core where theyarestoreduntilreleased.Itwasseenearly ends oftheprocessesclosetoplasmamembrane, the TGN,ACTHvesiclesmustbetransportedto live cellimaginginAtT20cells. interesting questionandhasrecentlybeenstudiedusing transported fromtheTGNtotheirreleasesitewasan 2010, upon stimulation(reviewedelsewhere;Kogel&Gerdes and storage,thenfusionwiththeplasmamembrane many proteinsinvolvedinthematurationofvesicle on thematuregranule.Indeed,vesiclecoatcontains structures indicativeoftheclathrintriskelionwerefound during thegranulematurationprocessasnovisible clathrin coat (Tooze & Tooze 1986), which was removed into condensingvacuolesthatwereseentocontaina At theTGN,EMstudiesshowedthatPOMCwassorted Transport andexocytosisofPOMCsecretory vesicles granule formsandmatures. sorting and retention of the prohormone as the immature initial budding,andthisinteractionresultsintheactive al.2013),atthelumenalsideofTGNduring et candidates, in addition to SgII (Sun and SgIII are primary multiple m the granules of the RSP likely requires interaction with DOI: 10.1530/JME-15-0323 http://jme.endocrinology-journals.org Thematic Review In theAtT20cells,afterinitialsiteofbuddingat . 2013).HowPOMCvesiclesare Bonnemaison et al embrane-associated molecules,ofwhichCPE n

cawley andothers Printed inGreatBritain

i. 5). cleavage atsignaturepairs ofbasicresidues(Fig. range. ThefirststepinPOMC processingisendoproteolytic maturation ofPOMChave tofunctionwithinthispH various endoproteases and exopeptidases involved in the . 1984).Hence,theprocessingenzymes thatinclude et al granulepHisbetween4.5 and5.5(Loh the secretory In theTGN,pHis~6.8(Seksek granule. cleavages occurswithintheimmaturesecretory siteofproteolytic begin attheTGN,althoughprimary convertases Prohormone POMC processing atpaired basicresidue-specific sites maturation ofPOMCanditsderivedpeptides. processing stepsandtheenzymesinvolvedin 5).Inthissection, we describethesequential (Fig. et al. 2013). TGN throughinteractionwithperi-GolgiF-actins(Lou suggestive ofaroleinPOMCvesiclebuddingfromthe accumulation ofPOMCvesiclesattheTGNinAtT20cells a C-terminal(CT)tailregionofγ-adducinalsocausedan a storagezoneforthematuregranules.Overexpressionof CPE C-terminuscan interact with as thegranulearrivesatendofmicrotubules, just undertheplasma membrane. Itwasproposed that 2010), aproteininvolvedinthecorticalactinassembly . two-hybrid studieswasidentifiedasγ-adducin(Louet al additional interactingproteinelucidatedfromtheyeast- the vesicleanchorwasthroughC-terminusofCPE.An microtubules bythemotorproteins,kinesins2and3, POMC vesicleswereanterogradedlytransportedalong (Park inhibit endogeneousCPEC-tailinteractionwithdynactin the cytosoltoactasadominantnegativemolecule C-terminus of CPE was constitutively overexpressed in vesicles inAtT20cellsthatwaseliminatedwhenthe bylivecellimagingofPOMC-RFP-containing observed vesicles atarateof~0.5µm/s(Scholey2013),speed transport, respectively. Interestingly, kinesin 2 transports dependent motorproteinsforanterogradeandretrograde contained kinesins2and3aswelldynein;microtubule al.2008).Thecomplex from AtT20celllysates(Parket that theC-terminusofCPEspecifically boundtodynactin α-MSH (Ac-ACTH(1–13)-NH enzymes, togenerate the POMCpeptidessuchasACTH, endoproteases, exopetidases, acetylation, and amidation Processing ofPOMCinvolvesmanyenzymes,including Processing ofPOMC peptides Biosynthesis ofPOMC-derived Published byBioscientifica Ltd. t al.2008).Theseresultsdemonstratedthat et Downloaded fromBioscientifica.com at09/29/202106:18:27PM 2 ), Processing ofPOMCcan β-LPH, and γ-adducin to establish . 1995),whereas et al 56 : 4 β-endorphin T82 via freeaccess

Journal of Molecular Endocrinology . 1993 al, its pro-segmentcleavagesite (Benjannetet activation onthecarboxyl sideoftheRSKRmotifin a pre-Golgicompartmentsuggestive ofanautocatalytic fully processed.Theactivation ofPC1/3beginsearlyin to workinthematuregranule whereprohormonesare al.1994, et function atanacidicpH(Zhou&Lindberg1993, processing occurs.Thematureformsoftheseenzymes granules oftheRSPwheremajorityPOMC pro-form and arespecifically trafficked tothe secretory 2011, Chretien&Mbikay2016). ofthe PCs,seeSeidah review ofthehistoricaldiscovery prohormones atspecificpairedbasicresidues.(For a al. 1991) and other shown to cleavePOMC (Thomas et related toyeastkexinandwereclonedin1991 are PC1/3 and PC2. These PCs are subtilisin-like enzymes The majorendoproteolyticenzymesforthesecleavages humans maynotoccurnaturally(Scott&Lowry1974). -END, -endorphin;JP, joiningpeptide;-MSH,-melanocyte-stimulatinghormone;PC,prohormoneconvertase;Ac,acetyl;K,lysine;R,arginine.*α-MSHin B-like; AACE,acidicACTH-convertingenzyme;CPE,carboxypeptidaseE;NAT, N-acetyltransferase;PAM, peptidylglycineα-amidatingmonooxygenase; ACTH, andβ-LPH.AmorecomprehensiveprocessingpatternisseenintheILhypothalamus,yieldingα-MSH-endorphin.APB,aminopeptidase General schematicdepictingtheprocessingofbovinePOMC.InAL,PC1/3isprimaryconvertaseinvolvedingeneration16-kDaN-POMC, Figure 5 http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0323 Thematic Review Both PC1/3andPC2enzymesaresynthesizedasa hypothalamus Intermediate lobeand NH NH Anterior lobe 2 2 POMC(1–49) e al.1995);hence,theyareideal Lindberg et PC2, Y

APB 16k N-POMC( apsin A L ys- γ 3 –MSH γ RK 3 RK -MSH n

1–77) cawley andothers JP Printed inGreatBritain CPE/P Ac KR KR NA

ACTH(1–14) T ACTH α AM Friedman -MSH(1–13) JP (1–17) AACE, PC2 CPE PA PC1 POMC KR M

CLIP (ACTH(18–40)) APB CLIP (ACTH(19–39) KKRR KKRR activity onothersubstrates. InAtT20cells,proSAAS al.2000),andthereforeregulatesits for PC1/3(Qianet PC1/3 toyieldapolypeptide thatactsasaninhibitor al.2000)hasbeenfoundtobeprocessed by (Fricker et inhibitor ofPC1/3(Leeet al . 2004).Additionally, proSAAS peptide expressedintransisabletoactasanendogeneous control ofendogeneousinhibitors/chaperones.PC1/3pro- the activityofboththesePCsappearstobeunder β-endorphin, respectively(Zhouet al. 1993).Interestingly, ACTH, andβ-LPHtoyieldN-POMC(1–77),α-MSH, be responsibleforthecleavageof16-kDaN-POMC, granule andfoundto later intheimmaturesecretory granules. PC2isactivated within theimmaturesecretory in thiscompartment, although themajor cleavage occurs POMC toyield16-kDaN-POMC,ACTH,andβ-LPH(Fig. 5 ) in theGolgi,itcanbegintoactonfirstcleavagesof Because the ~87-kDa form of PC1/3 is active and present Zhou &Lindberg1993, amide peptides Biosynthesis ofPOMC-derived ACTH Published byBioscientifica Ltd. PC1 KR CPE γ -lipotropin ) PC2/? KK KK Downloaded fromBioscientifica.com at09/29/202106:18:27PM KK β -lipotropin * Goodman &Gorman1994). β β -MSH -MSH PC2, Y Ac CPE KR KK NA apsin A T β β -END(1–31) -END(1–29) β 56 -END(1–27) : 4 KK KK K PC2/? CPE COOH T83 via freeaccess Journal of Molecular Endocrinology CT peptideatVVAKK granules.PC2inturncleavesthe immature secretory processed into PC2 withinthe acidic environment of granulewhereitisautocatalytically immature secretory acts asapotentinhibitor. Pro-PC2isthensortedintothe The CTpe peptide. CT where 7B2 is cleavedbyfurin to generate a 31-aminoacid with 7B2intheERandisthentraffickedtoGolgi . 1997).Pro-PC2formsacomplex activation (Mulleret al chaperone tohelpwithitstransporttheGolgiand the case of pro-PC2, a p POMC underpulse/chaseco expression hasbeenfoundtoinhibittheprocessingof . 1996). Paquet et al experiments (Zhou subsequently affirmed by additional ): (Fig. 5 observations andhypothalamicneurons intermediate pituitary β-LPH toyield-endorphininmelanotrophs inthe to yieldN-POMC(1–77),ACTHα-MSH,and to PC1/3, whereas PC2 cleaved 16-kDa N-POMC corticotrophs ACTH, andβ-LPHinanteriorpituitary at pairedbasicresiduestogenerateN-POMC(16 Mains & Eipper 1981b) attributing cleavage of POMC hypothalamic neurons (Loh 1979, cellsand cultures ofanteriorandintermediatepituitary vitro pulse–chase studies using AtT20 and primary This ledtofurtherunderstandingoftheprevious in al . 1991). thatsynthesizesα-MSH(Seidahet pituitary it wascolocalizedwithPC2intheintermediate corticotrophsthatsynthesizeACTH,whereas pituitary PC1/3 mRNAwasexpressedprimarilyinanterior included processing camefromseverallinesofevidence.This in thesecells(Bergeronet al.2002). reflecting apossibleroleinPOMCprocessingandsecretion cells isnegativelycorrelatedtocellularlevelsof7B2,also 2001). Indeed,stimulatedsecretionofACTHfromAtT20 also insecretionofitsderivedpeptides(Mbikay role of 7B2 not only in POMC processing indirectly, but normally produce lack ofprocessing by an active PC2 enzyme that would duetothe secretion ofACTHfromtheILpituitary die earlyduetoCushing’s diseasebecauseofexcessive the 7B2KOmouse.Thesemice,unlikePC2 The importanceof 7B2 in POMC processing is seen in . 1999, et al PC2 enzyme(Fortenberry inactive formoftheCTpeptidethatliberatesactive basic residues KK by the exopeptidase, CPE, to generate an DOI: 10.1530/JME-15-0323 http://jme.endocrinology-journals.org Thematic Review The physiologicalimportanceofPC1/3inPOMC in situhybridizationstudies,revealingthat α et al. 1993, In vivostudiesusinggeneKOinmice -MSH in this tissue. This indicates a 189↓ rotein named 7B2, is required as a ptide then binds to pro-PC2 and ptide thenbindstopro-PC2and SVP, followed byremovalofthe nditions (Lee Friedman n

cawley ). . 2001). Mbikay et al e al. , 1980 Liotta et et al. 1994,1996, andothers Printed inGreatBritain . 2004).In et al kDa), t al. et

. 1996).Yapsinneuroendocrine tissues (Cawley et al 1-like yapsin 1-likeproteinsinbovine andmouseendocrine 1 hasbeenusedtoimmunologically identifymammalian optimum of4.0–5.0.Anantibody generatedagainstYapsin been characterizedasan~70-kDa enzymethathasapH . 1993).Yapsin et al Ahas basic residuesaswell(Azaryan al.1990).Yapsin 1isabletocleavePOMCatpaired et residues, similartotheyeastkex-2enzyme,(Egel-Mitani been showntoprocesspro-α-matingfactoratpairedbasic 3, ageneproductoftheyps1 This enzyme is related to Yapsin 1 or yeast aspartic protease ; β-endorphin (Fig. 5 basic residuestoyieldN-POMC(1–77),ACTH,β-LPH,and granules. IthasbeenshowntoprocessPOMCatpaired granulesaswellfromadrenal chromaffin secretory ILandneurallobe homogeneity frombovinepituitary an asparticprotease,hasbeenpurifiedtoapparent enzyme knownasYapsin A(orPOMC-convertingenzyme), Yapsin A in vivoPOMCprocessing. current findingpointstoanimportantroleofPC1/3 PC1/3 nullmicethatarenotobese(Zhu glucose homeostasis.Althoughthispatientdiffersfrom (Jackson of partially processed POMC intermediate ACTH products of theenzymeandhighcirculating levelsofseveralforms mutation in the PC1/3 proteinduetobothsplicingandnonsynonymous (Friedman and ACTH(1–17))aswellβ-endorphinLys-γ-MSH to ACTH-relatedpeptides(ACTH(1–14),ACTH(1–15), exogeneously expressedPOMCwascompletelyprocessed GH3 cellsthatexpressPC2andnotPC1/3,showing always true.Thiscorroborateswithinvitro studiesin often compensatedforbyPC2,butthereverseisnot al.2010)KOmicerevealedthatthelossofPC1/3is et analyses ofPC1/3(Wardman. 2010)andPC2(Zhang et al POMC-derived peptides(Furuta show retardedgrowth,andtheydonotfullyprocess prohormones. PC2nullmicelooknormalatbirthbut because theenzymeisresponsibleforprocessingother also exhibit retarded growth and developmental defects yet havenormallevels of circulating corticosterone. They null miceprocessPOMCpoorlytoACTH(Zhu these enzymesarenot essential forlife.Moreover, PC1/3 (Jackson et al. 1997, the findingoftwohumanpatientswithdefectsinPC1/3 for PC1/3andPC2(Furutaet al. 1997, peptides Biosynthesis ofPOMC-derived Published byBioscientifica Ltd. . 1997).Thepatientwasobeseandhadpoor et al

t al. ). 1996 A female patient deficient in et In addition to PC1/3 and PC2, another PC1/3 gene showed low expression levels Farooqi et al. 2007)showedthatboth . 1985, Loh et al Downloaded fromBioscientifica.com at09/29/202106:18:27PM gene inyeastthathasalso t al.1997).Peptidomic et . 1995) et al Azaryan Zhu et al. 2002)and 56 : 4 . 2002),the et al et al. 2002) T84 via freeaccess

Journal of Molecular Endocrinology extended lysineandarginine residues,CPEwasconsidered processed, andbecauseofits specificityforC-terminally granules, wherepeptide hormone intermediatesare optimum activityintheacidic environmentofsecretory Kanmera &Chaiken1985).Becauseofitslocalizati were alsoshowntoberemovedbyCPE(Hook&Loh1984 , and arginineresiduessuchasvasopressinoxytocin time, otherpeptideswithC-terminallyextendedlysine ACTH(1–15), andACTH(1–14)(Hook&Loh1984).Atthat liberated byPC1/3fromPOMC,togenerateACTH(1–16), remove thebasicresiduesfromACTH(1–17),apeptide specifically inhibitedbyGEMSA.Itwasalsoshown to has apHoptimumof5.5thatisstimulatedbyCo is ametalloproteasewithZnboundattheactivesite. It peptides (Fricker&Snyder1982, extended lysineandarginineresiduesfromenkephalin 1982 as an enzyme capable of removing C-terminally carboxypeptidase B-like enzyme) was first discovered in H (initiallyknownasenkephalinconvertaseor biologically activepeptides.CPEorcarboxypeptidase is required to remove the CT basic residues to yield the cleavage of POMCatpairedbasicresidues, an exopeptidase Carboxypeptidase E/H Exopeptidases inPOMCpeptideprocessing processing ofACTHtoα-MSHbesidesPC2. has not been cloned, but AACE could play a role in the cleave thepairedbasicresiduesofPOMC.Theenzyme corticotropin-like intermediatepeptide(CLIP)butdidnot residues oftheArg . 1992).AACE-cleavedACTH(1–39)atthetetrabasic et al granules (Estivariz to be present in bovine IL secretory highly specific fortetrabasic residues, has beenreported enzyme (AACE),withapHoptimumof5.0–6.0andbeing activated serineprotease,namedacidicACTH-converting Tetrabasic residue-specificenzymes produce aphenotype. such thatgeneticdefectsinthePCsmaynotnecessarily in ensuringthatprohormoneprocessingismaintained, . Additional ‘backup’ enzymes could be important in vivo a roleinprocessingofPOMCandotherprohormones endocrine tissue,similartoYapsin 1inyeast,mayplay suggest thatamammalianasparticproteasepresentin Although Yapsin Ahasnotbeencloned,currentstudies by processingproglucagon islet IR hasalsobeenfoundexclusivelyinhumanpancreatic http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0323 Thematic Review α-cells, andpurifiedyapsin1cangenerateglucagon 17 –Arg 18 bondtoyieldACTH(1–17)and Subsequent to endoproteolytic Subsequent toendoproteolytic e al.2011). in vitro (Cawleyet n Hook

cawley . 1982a).CPE et al andothers Printed inGreatBritain

A calcium- on and 2+ and the CPE-deficientmouse,Cpe from POMC.Indeed,proteomicanalysisofpituitaries peptide hormoneintermediates,includingthosederived carboxypeptidase for most if not all to be the primary elevated inthecirculation ofCpe years.POMClevelswere mice withintheintervening (Hosaka & Loh1997),afindingnotreproducedlaterbyothers stimulation bycorticotropin-releasinghormone(Shen constitutively athighlevels,showingnoresponse to (Shen &Loh1997).Furthermore,POMCwassecreted ACTH, althoughlarger24-kDaformofACTHwaspresent in theanteriorpituitary, and itwaspoorlyprocessedto POMC wasaccumulated24-foldabovenormalanimals t al.1995).IntheseCpe et with severeobesity, diabetes,andinfertility (Naggert gene wasfoundintheCpe evident fromseveralstudies.AmutationintheCpe enzyme and asorting receptor for prohormones was see prevention ofstress-induceddepression(forareview, molecule actingextracellularlyinneuroprotectionand other importantnonenzymaticrolesasasignaling CPE issecreted,andseveralstudiesindicatethatitplays residues frompeptideproductsliberatedPOMC. granule, whereitcanactenzymaticallytocleavebasic form (molecularweight~50kDa)withinthesecretory of theCPEisthenprocessedtomaturesoluble granulesafterbudding.Someinto immaturesecretory interaction with lipidraftsandis subsequently sorted where itassociateswiththemembranethrough (pro-CPE, ~55kDainsize),istraffickedtotheTGN vesicles’ section). CPE, synthesized as a precursor (see the‘Transport andexocytosisofPOMCsecretory transport byassociatingwithmicrotubulemotors granuleand thecytoplasmictailisinvolvedinsecretory act asasortingreceptorforprohormonesattheTGN, et al. 2002, membrane givingrisetoacytoplasmictail(Dhanvantari granulea transmembraneorientationinthesecretory ( enzymatically muchmoreactivethanamembraneform in vivo(Cheet al.2005). the roleof CPE inthenormal processing of thesepeptides derived peptides,comparedwithWTpituitary, indicating accumulation oftheCTbasicresidue-extendedPOMC- inefficient sortingofPOMC intothegranulesofRSP mice. This poor maturation of POMC could be aresult of peptides Biosynthesis ofPOMC-derived Hook 1985).Someofthemembraneformscanassume Published byBioscientifica Ltd. Cawley The physiologicalimportanceofCPEasaprocessing There are two forms of CPE: a soluble form that is t al.2005),possibly reflecting a change in the et Zhang et al. 2003).Themembraneformcan et al. 2012). Downloaded fromBioscientifica.com at09/29/202106:18:27PM fat/fat fat/fat mice,itwasreportedthat fat/fat fat/fat mousethatpresented , showedasignificant miceversusnormal 56 : 4 T85 via freeaccess

Journal of Molecular Endocrinology in the POMC-FOXO1 neuropeptides, level. Consequently, increased levels of thehypothalamic FOXO1 isacorepressorofCPEexpressionatthepromoter affecting energyexpenditure.Thestudyshowedthat residue doubletsbyYapsin Ahasalsobeen reported.Thus, two basicresidues.Cleaving ofPOMCinbetweenbasic arginine indicatesthatcertain cleavagesoccursbetween residues byPC2toreleaseCLIP (Fig. 5) withanN-terminal paired basicresidues,thecleavage ofACTHatthetetrabasic cleave POMCandderivedpeptidesonthecarboxylside of Aminopeptidases processing andsorting. emphasizing thecriticalroleCPEplaysinprohormone al.2015),further as intellectualdisability(Alsterset patient presentedwithobesity, type2diabetes, aswell homozygous nullmutationforCPE,whichshowedthe recent description of the first human witha truncating of CPEinobesity. Corroboratingthesemouse studiesisa f . 1996).Thisstudy convertases’ sectiononPC2;Zhuet al ACTH(1–16) intermediatetoα-MSH(seethe‘Prohormone Concomitantly, theCPEcanprocessacetylated by inactivationoftheCTinhibitorpeptide7B2. that subsequently affected the levels of active PC2 expression ofCPEinthePOMChypothalamicneurons neurons (POMC-FOXO1 ablation ofForkheadboxproteinO1(FOXO1)inPOMC that balance. Insupportofthisweretheobservations CPE played a key role in appetite regulation and energy increased foodintakeandobesity, demonstratingthat peptide thatcontrolssatietyinthistissue,resulted of hypothalamicPOMCtoα-MSH,amajoranorexigenic al.2004).Inbothcases,defectiveprocessing (Cawley et in thehypothalamiccircuitry thatcontrolssatiety contributed inpartbyautophagyduetoadisruption observed phenotypeintheCpe observed phenotype intheseanimals(Srinivasanet al.2004). residues thatwereinactive,resultingintheinfertility al.1995)andGnRHpeptideswithextendedbasic et 1997). Thesemicealsohadhyperproinsulinemia(Naggert POMC productsthataccruedintheGolgi(Shen&Loh resulting inconstitutivesecretionofpartiallyprocessed for fullprocessingbecauseCPEactsasasortingreceptor, FOXO1 peptide, obesity paradigm, by increasing the satiation POMC protected the mice against weight gain, in a diet-induced urther demonstrates the important physiological function DOI: 10.1530/JME-15-0323 http://jme.endocrinology-journals.org Thematic Review As indicatedpreviously, obesity was also an −/− . 2009).Hence,inthisPOMC- α-MSH (Plumet al model, deletionofFOXO1allowedincreased -endorphin, were observed α-MSH andβ-endorphin,wereobserved −/− Although PC1/3andPC2generally mice. FOXO1 deletion therefore −/− ) reducedfoodintakewithout fat/fat n

cawley andCPEKOmice, andothers Printed inGreatBritain

has apHoptimumof6.0,isstimulatedbyCo activity isfoundasbothasolubleandmembraneform, . 1984).Theenzymatic granules(Gaineret al NL secretory ILand B-like enzymehasbeenfoundinbovinepituitary remove theN-terminalbasicresidue.Anaminopeptidase there isaneedforanaminopeptidaseB-likeenzymeto and Ac-β-endorphin,respectively. Thisenzymatic ACTH(1–14) andβ-endorphintoyield N-Ac-ACTH(1–14) is responsible for acetylating the N-terminus of both granules indicate that the same acetylation enzyme activities (Chappell studies ofACTHandβ-endorphinacetylationenzymatic granules,aswell ascomparative from bovinesecretory β-endorphin peptidesandacetylationenzymaticactivity (Glembotski 1982) using fragments of ACTH and Gibson &Glembotski1985).Competitionstudies al.1982, β-endorphin (Chappell et granules thatcouldacetylatebothACTH(1–14)and secretory found inbovineandratintermediatepituitary to <5%inadultrats(Alessiet al.1983). P14, butintheAL,N-Ac-β-endorphindropsdramatically the levelincreasesto90%ofendorphinspresentby IL frompostnatalday1(P1)throughadulthood.IntheIL, N-Ac-β-endorphin hasbeenfoundinboththeALand amongspecies. existence oftheselatterformsdovary IL,although the forms arealso present inthepituitary the predominantform,diacetylatedanddeacetylated While ithasbeenreportedthatinmammals,α-MSHis the relative amounts of melanotropic and opiate activities. derived from POMC, acetylation could be used to regulate activity (Deakinet al. 1980).Becausethesetwopeptidesare acetylation ofβ-endorphincompletelyabolishesitsopiate potentiated and its half-life increased by N-acetylation, activity ofα-MSH(Guttmann&Boissonnas1961)is effects on the POMC peptides. Although the melanotropic circulation. Acetylation also has profound biological of aminopeptidasesandenhancetheirhalf-lifeinthe of thepeptidebyprotectingthemagainstaction toincreasethestability peptide hormonesmayserve Acetylation ofPOMCpeptides Acetylation andamidationofPOMC-derivedpeptides granules(Castroet al.1989). same secretory secreted withα-MSH,indicatingitscolocalizationinthe that itisan~70-kDaglycoproteinandcoordinately such as in CLIP. Characterization of the enzyme indicates not cleaveanN-terminalArgifitisfollowedbyaproline and cleavesArgpreferentiallyoverLys. However, itwill peptides Biosynthesis ofPOMC-derived Published byBioscientifica Ltd. An N-acetyltransferase enzymaticactivityhasbeen ) from rat IL secretory al.1982)fromratILsecretory et Downloaded fromBioscientifica.com at09/29/202106:18:27PM 56 Glembotski 1982, N-acetylation of : 4 2+ andZn T86 2+ via freeaccess ,

Journal of Molecular Endocrinology the anteriorandintermediate pituitary, andthearcuate POMC isdifferentiallyprocessed endoproteolyticallyin Regulation ofPOMCprocessing in thisbookissue. PAM arereviewedbyMainsandEipper(Kumar (PAM). StudiesontheamidationofPOMCpeptidesand reaction ispeptidylglycineα-amidatingmonooxygenase is alsoamidated.Theenzymeinvolvedintheamidation 5) another POMC-derived peptide, joining peptide (Fig. involves theamidationofN-Ac-ACTH(1–14).Inaddition, Amidation of POMC peptides mechanism fortheregulationoftheirhormonalactivity. acetylation ofPOMC-derivedpeptidescontributesanother normally releasedundernonstressedconditions.Thus,the form releasedisβ the anteriorpituitary, afterrepeatedstress,themajor al.1985).Bycontrast,in major forminplasma(Akilet of stress selectivelyincreasedthebiosynthesisandrelease activity.their secretory Ithasbeenreportedthatrepeated that acetylationofthesepeptidesmaybemodulatedby antagonists (Ham&Smyth1984).Thesestudiessuggest in culturewhenexposedtodopamineagonistsand of in theRERorGolgi.Additionally, changesinacetylation secretogogue activitiesbutnottheacetylationactivity granuleswasaffectedbythesepro-andanti- secretory al.1986).Onlytheacetyltransferaseactivityfrom et a dopamineagonist,hadoppositeeffects(Millington levels, andPOMCpeptides,whereasbromocryptine, increased theacetyltransferaseactivity, POMCmRNA that haloperidol,adopamineantagonist,coordinately by dopamine(Fischer&Moriarty1977).Itwasfound control isunderinhibitory the intermediatepituitary ( been showntobecoregulatedintheintermediatepituitary if theyaresimilaroridenticalmolecules. havenotbeencloned,itremainstobedetermined pituitary acetylation enzymesinthebovineandratintermediate granule is inhibitedbydetergents.Becausethesecretory in thepituitary, OMAT hasapHoptimumof6.0–6.6and . 1982).Unlikeotheracetylationenzymaticactivities et al opiomelanotropin acetyltransferase(OMAT) (Chappell hasbeenreferredtoas of ratintermediatepituitary granules activity specificallylocalizedtothesecretory ). Secretion of POMC peptides from . 1986).SecretionofPOMCpeptidesfrom Millington et al http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0323 Thematic Review N β N -endorphin have been demonstrated in rat IL cells -endorphin havebeendemonstratedinratILcells -Ac-β -acetyltransferase activity and POMC expression have -acetyltransferase activityandPOMCexpressionhave -endorphin(1–31) from the IL of rats and is the -endorphin(1–31) fromtheILofratsandis -endorphin(1–31), ratherthanβ n

cawley Synthesis ofα-MSH andothers Printed inGreatBritain et al. 2015) -LPH, -LPH,

into N-POMC(1–49)andLys- γ tissue this POMCintermediatewascleavedinpituitary exemplifies such amechanismbecause only ~50% of to cleavethesite.Indeed,fateofN-POMC(1–77) cleavage activesitecaninfluencetheabilityofenzyme differential processing. 5).Anumberoffactorscandictatethe products (Fig. nuclei ofthehypothalamustogeneratedifferentend next. Thissectionisnotintended asacomprehensivelist peptides is regulated by various secretagogues as discussed from thehypothalamus.The secretionofPOMC-derived β-endorphin areprimarilysecreted bymelanotrophsand are secretedbycorticotrophs, whereasα-MSHand For example,ACTH,β-LPH,andsome-endorphin neurons ofthearcuate nucleusofthehypothalamus. gland, respectively,the pituitary as well as from peptidergic from corticotrophsandmelanotrophsoftheALIL of secretagogues. POMC-derivedpeptidesaremainlysecreted granules andsecretedinresponsetosimulationby packaged andstoredaselectron-densecoresinsecretory prohormone convertases,POMC-derivedpeptidesare After posttranslationalprocessingviacleavageby Secretion ofPOMC-derived peptides et al. 1994,Joshi1995). processing ofACTHtoα-MSHinthesesneurons(Zheng hy β-LPH inthesetwolobes.ThepresenceofPC2the contributes tothedifferences in processingofACTHand (Estivariz in theALofpituitary AACE ispresentinmuchhigheramountstheILthan vivo. Additionally, thetetrabasic residue-specific enzyme primarily PC1/3 that does not catalyze theses cleavages expresses β-LPH to-endorphin,theanteriorpituitary PC2 resultingintheprocessingofACTHtoα-MSHand expressesPC1/3and Whereas theintermediatepituitary of differentprocessingenzymesasreviewedpreviously. of POMCintheALandILisdictatedbypresence activity itexhibits(Pepper&Bicknell2009). controls thelevelofN-POMC(1–49)andmitogenic because theregulationofprocessingthatthissiteproduces processing. Thishasimportantphysiologicalsignificance stearic hindrancebythesugarmoietycouldprevent into N-POMC(1–49)(Bennett1986),suggestingthat Thr-45 regulatedtheprocessingofN-POMC(1–77) analysis identifiedthattheO-linkedglycosylationof 1986). MetaboliclabelingstudiesandPOMCfragment peptides Biosynthesis ofPOMC-derived Published byBioscientifica Ltd. pothalamic POMCneuronscouldalsoaccountforthe Tissue-specific glycosylation ofaresidueclosetothe Another mechanismfortissue-specificprocessing Downloaded fromBioscientifica.com at09/29/202106:18:27PM 3 -MSH (Seger&Bennett . 1992),andthis et al 56 : 4 T87 via freeaccess in Journal of Molecular Endocrinology to thenetsecretionofPOMC-derived peptidesfromthe many other hormones and compounds that contribute negative regulationofglucocorticoids, althoughthereare primarily underthepositive regulationofCRFandthe of circulating ACTHundercontrol.Hence,theAL is the ALandhypothalamicneuronstokeeplevels negative feedbackinhibitiononthecorticotrophs in physiological roles,glucocorticoidsinturnexhibit a in thebodyresponsetopain.Amongtheirmany β-LPH/-endorphin isreleasedtoactivateopioidpathways glucocorticoids duringtimesofstress.Inaddition, which inturnactivatestheadrenalcortextoproduce viathehypophyseal vein, The ACTHexitsthepituitary al.1982). the AL,includingprimarilyACTH(Chanet of POMC-relatedpeptidesfromthecorticotrophs (Swanson et al. 1983),whichthenstimulatesthesecretion also knownasCRH)intothehypophysealportalsystem PVN secretecorticotroph-releasingfactor/hormone(CRF, term orlong-termstress,theneuronsinhypothalamic the ALaffectsecretionfromILtoo. proximity totheAL,moleculessecretedintoandthrough hormones fromcellsoftheAL.BecauseILisinclose network, whichinturnregulatesthesecretionofpeptide neurotransmitters andpeptidehormonesintotheportal in thelowerinfundibularstemclosetoALandrelease network from thePVNterminateearlieratcapillary the circulation throughthe hypophysealvein.Otheraxons bedoftheposteriorlobeandarereleasedto the capillary nucleus (SON)andparaventricular(PVN))innervate the magnocellularneuronsofhypothalamus(supraoptic containing argininevasopressin(AVP) andoxytocinfrom stalk.Axons throughthepituitary stem tothepituitary extend throughthemedianeminenceandinfundibular fibersfromthehypothalamus and reproduction.Nerve with multipleendocrinefunctionssuchasgrowth,stress, axisandisinvolved the hypothalamic–pituitary–adrenal 6).Itisthemiddlecomponentof hypothalamus (Fig. neural lobeistechnicallyconsideredanextensionofthe by manytobethemasterendocrinegland,although present betweentheALandNL,isconsidered lobe (neurohypophysis,orneural(NL))withanIL composed oftheAL(adenohypophysis)andposterior from theALofpituitary Regulation ofsecretionPOMC-derivedpeptides pathways thatplayrolesinthisprocess. ofseveral exceedingly complex,butmoreasasummary of effectorsPOMC-derivedpeptidesecretion,asthatis DOI: 10.1530/JME-15-0323 http://jme.endocrinology-journals.org Thematic Review With respect to POMC, in vivo, in response to short- n

The pituitary gland is gland is The pituitary cawley andothers Printed inGreatBritain neurons mayregulateandcontributetothereleaseof secretion ofβ-LPH homolog, wasshowntoreducetheserotonergic-induced corticotrophs. Indeed, dexamethasone, a glucocorticoid secretion ofPOMC-derivedpeptides fromthesetissues. for otherneurotransmittersandpeptide hormonesthathelpregulatethe of thehypothalamustoreleaseα-MSHand β-endorphin. Seemaintext secreted fromadipocytesactivatesPOMC neuronsinthearcuatenucleus α-MSH andβ-endorphinfromthemelanotrophs of theIL.(B)Leptin manner. Dopamine(DA)inhibitsandCRFincreasesthesecretion of then inhibitACTH,β-LPH,and-endorphinreleaseinanegativefeedback the secretionofglucocorticoidsfromadrenalcortex.Glucocorticoids hypothalamus duringtimesofstress.ThesecellsreleaseACTHthatcauses in theALareunderpositiveregulationbyCRFreleasedfrom Simplified schematicofthehypothalamic–pituitaryaxis.(A)Corticotrophs Figure 6 peptides Biosynthesis ofPOMC-derived B A Published byBioscientifica Ltd. AL HYPOTHALAMUS (Arcuate nucleus) Glucocorticoids Corticotrophs LEPTIN STRESS HYPOTHALAMUS (PVN, SON) CRF in vivo,suggestingthatserotonin NL DA Downloaded fromBioscientifica.com at09/29/202106:18:27PM α β IL -MSH, -END α 56 β -MSH, -END β β ACTH, : 4 -END -Lipotropin Pituitary T88 via freeaccess Journal of Molecular Endocrinology ( e.g. melatoninandbonemorphogeneticprotein 4 affects thecellularcontent and hence secretion profiles, al.1997) synthesis ofPOMCbysecretagogues(Aokiet al.2013).Indeedtheregulationofexpressionand et CRF-stimulated ACTHproductionandsecretion(Shin activities, and intracellular cAMP levels, thus augmenting increased basalandCRF-stimulatedPOMCpromoter andhypothalamusafterstress, secreted fromthepituitary secretion via effects on CRF, e.g. clusterin, a peptide many otherfactorshavebeenshowntostimulatetheir negative regulationofPOMC-derivedpeptidessecretion, et al. 1986). on theCRF-regulatedcorticotrophsecretion(Petraglia role and CRF neurons, and exerts a tonic inhibitory through aninteractionbetweenGABAergicneurons (GABA) canalsoinhibitβ-endorphinsecretion,possibly of POMC-derived peptides. Gamma-aminobutyric acid This wasconsideredanexampleofdissociatedsecretion specifically expressingβ-endorphinandnotcorticotrophs. tissue regulation onpituicytesorothernonpituitary reduced levels.Alternatively, theeffectcouldbethrough POMC toβ-endorphin in corticotrophstoaccount for the with somatostatin possibly reduced the processing of confounding; however, itwasproposedthattreatment β-endorphin the selective inhibition of CRF-induced secretion of . 1997).Forsomatostati not supportthis(Bowmanet al was throughcGMPsignaling;however, laterstudiesdid function. ForANF, ithadbeenproposedthatitsinhibition inhibit POMC-derivedpeptidesecretionbyaffectingCRF al.1991),havebeenshownto somatostatin (Invittiet . 1986)and atrial natriureticfactor(ANF)(Shibasakiet al corticotrophs invivo(Sapun-Malcolmet al.1983). β-LPH (andbyassociationACTH)fromanteriorpituitary angiotensin systemincreased thesecretionofACTH, . 1989).Inaddition,therenin– kinase C(Fagarasanet al enhanced secretionofβ from cells of the immune system in response to infection, & Gerhards 1987). Interleukin-1, a cytokine released independent ofthephospholipaseA2activation(Knepel β rat ALresultinginthestimulatedsecretionofACTHand phospholipase A2,generatedasignalincorticotrophs of component ofbeevenomandapowerfulstimulator of mechanisms. Forexample,Melittin,themajorpeptide viadiverse related peptidesfromtheanteriorpituitary compounds alsoappeartostimulatesecretionofPOMC- e al . 2010, Tsukamoto et -endorphin, althoughthemechanismappearedtobe http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0323 Thematic Review In contrasttothosemoleculesinvolvedinthe In additiontoglucocorticoids,otherfactors,suchas in vivo,andnotACTHβ-LPH,was -endorphin byinducingprotein 2013). Somehormonesor n

cawley andothers Printed inGreatBritain n,

high levelsofK to secretagoguesorbymembranedepolarizationwith peptides inacalcium-dependentmannerresponse store and secrete ACTH- and β-endorphin-related POMC-derived peptides.Thesecells,asnotedpreviously, trafficking aswellintheregulationofsecretionits extensively inthestudyofPOMCbiosynthesisand POMC maturation’section, AtT20 cells have been used in thesepathways. these immunosuppressantdrugsactatacommonpoint stimulated secretion(Sheppard1995),demonstratingthat peptide secretion and potentiated phorbol ester- and CRF- immunosuppressant drugs,stimulatedPOMC-derived molecules suchascyclosporinAandtacrolimus(FK506), to ACTH affects corticotroph behavior. Other small . 1984).Hence,regulationofadrenalresponsiveness et al glucocorticoid secretion from the adrenal cortex (Costa gland by reducing release from the anterior pituitary responsiveness toACTH,mightincreaseβ-endorphin POMC, nimodipine, which reduced adrenal gland on theregulationofbiosynthesisandsecretion that, becauseglucocorticoidsexhibitfeedbackinhibition through anactionontheadrenalglands.Itwasproposed antagonist, nimodipine,increasedβ-endorphinsecretion thatthecalcium peptides fromtheAL.Itwasobserved levels ofcontrolonthesecretionPOMC-derived CRF (Chan cells;however,anterior pituitary itwasnotaspotent culturesofhuman POMC-derived peptidesfromprimary 1982). AVP had been shown to induce the secretion of thus increasingthesecretionofβ effectontheAL, hypothalamus toexertitsstimulatory of AVP fromneuronsoftheSONsandPVNs β 16-kDa N-POMC from AtT20 cells, an effect that was fully stimulated thereleaseofACTH, β-endorphin, norepinephrine, amember of thecatecholaminefamily, activity.from thoseresponsibleforbasal secretory Indeed, underlying stimulatedhormone secretionweredifferent secretion in thesecells, indicating that the mechanisms potential activityhadnoeffectonbasalhormone in AtT20cells. However, acompleteblockade of action frequency andACTH/β-endorphin-likepeptidesecretion calcium concentrationsincreasedbothactionpotential nonselective in AtT20cells(Surprenant1982).Usingisoproterenol,a linked totheregulatedsecretionofmaturegranules potentials and calcium influx was shown to be closely peptides Biosynthesis ofPOMC-derived -LPH, andβ Published byBioscientifica Ltd. As indicatedinthe‘Intracellularorganizationof Small moleculestudieshavealsoidentifiedfurther et al. 1982). β-adrenergic agonist, or raising the external -endorphin bystimulatingthesecretion + . Membranedepolarizationbyaction Downloaded fromBioscientifica.com at09/29/202106:18:27PM -endorphin (Beuerset al. 56 : 4 β-LPH, and T89 via freeaccess

Journal of Molecular Endocrinology 3-deazaadenosine and also bythephospholipidmethyltransferaseinhibitors, selectively inhibits corticotroph secretion by dexamethasone,asyntheticglucocorticoidthat methylation. Theseeffectswereblockednotonly stimulated ACTHsecretionalsoincreasedphospholipid AtT20 cellsshowedthatCRFatconcentrations calcium signaling(Antoni1996).Otherstudiesin in CRFstimulationbyblockingtheCRF-dependent partly be due to a glucocorticoid-dependent reduction effectmay peptides (Sabol1980).Thefastinhibitory that inhibitsynthesisorincreasethecatabolismof by increasing transcription and translation of proteins the secretion of these peptides from these cells the corticotrophsinvivo,glucocorticoidsrapidlyinhibit secretagogues (Devi1992). of thegranulescontainingACTHinthesecellstomany CPE andACTH,demonstratingthewideresponsiveness converting enzymefoundinthesecells,parallelwith ionophore stimulatedthesecretionofadynorphin- forskolin, AtT20 cells,phorbolester, vasoactiveintestinalpeptide, regulated secretion (Mains & Eipper 1981a). Also in secretion wascalciumdependent,thehallmarkof blocked bycobalt,demonstratingthatthestimulated because most work on the IL has been done in tissue As such,therelevancetohuman physiologyislimited of thepituitary, oritisentirelyabsent(McNicol1986 ). thin layerofcellsbetweenthe ALandtheposteriorlobe in thefetusasadistinctarea butinadultsisreducedtoa melanocytes. However, inhumans,theILisonlypresent vein toaffecttheregulationofpigmentationby released intothebloodstreamthroughhypophyseal vertebrates haveawell-developedILinwhichα-MSHis from theILofpituitary Regulation ofsecretionPOMC-derivedpeptides above physiologicallevels(Mains&Eipper1981a when addedtotheculturemediumatup10,000times camel Furthermore, synthetichumanACTHand peptides (i.e.,ultrashortfeedback)wasfound. autoinhibition ofsecretionbyaccumulatedsecreted glucocorticoids ontheAtT20cells,noevidencefor secretion (Hooket al.1982b a CRFreceptor-mediatedeventassociatedwithACTH suggesting thatphospholipidmethylationmightbe DOI: 10.1530/JME-15-0323 http://jme.endocrinology-journals.org Thematic Review As withthefeedbackinhibitionbyglucocorticoidson In contrast to the negative regulation by β -endorphin didnotaltersecretionofpeptides β-adrenergic agonist,aswellthecalcium l -homocysteine thiolactone, ). n

cawley Mammals andlower andothers Printed inGreatBritain in vitro, but in vitro ).

by specifictonicinhibitionofdopamineD and secretionofPOMC-derived peptides fromthe IL and demonstratedatightregulationofPOMCexpression compounds didnotaffectCRFreceptorlevelsintheAL and antagonists (Shiver levels ofexpressioninresponsetodopamineagonists . 1987)butshowdifferent (Aguileraet al of thepituitary opposite. Indeed,CRFreceptorsarepresentonbothlobes bromocriptine, adopaminereceptoragonist,didthe al.1992),whereas CRF receptorexpression(Shiveret in rat IL (Hollt & Bergmann ) but 1982 decreased the addition, haloperidolincreasedPOMCmRNAexpression al.1989).In the dopaminereceptor(Kemppainenet D two howeverwereblockedbyhaloperidol(adopamine somatostatin, norepinephrine,andepinephrine;thelast and MSHfromdogILwasinhibitedbydopamine, signaling.Indeed,releaseofACTH and inhibitory net secretionwaslikelyabalancebetweenstimulatory AVP (Fischer&Moriarty1977),demonstratingthatthe se whereas othercompoundswerereportedaspotent the releaseofACTH-likematerialfromisolatedratNIL, response tohypothalamicsignals. peptides, inadditiontoβ-endorphin,thecirculation in melanotrophs, itsmainfunctionistoprovideMSH-like is composedprimarilyofahomogeneouspopulation at theinterfaceofneurohypophysis. Because theIL inaddition tobeing network ofthehypophyseal artery released fromthehypothalamicneuronsincapillary proximity to neurotransmitters and peptide hormones rat, anddogs.Asmentionedpreviously, theILisinclose obtained from a variety of animals, including frog, mouse, dopamine inthisvitro system.However, isoproterenol was presumablyduetothe lackoftonicinhibitionby ( in which these peptides were found in extracts of IL LPH-related peptideswere proportional totheamounts that theamountsofspontaneouslysecretedACTH-and melanotrophs tosecreteα stimulates corticotrophs to secrete ACTH and urotensin Iandsauvagine(Tran. 1990).Hence,CRF et al stimulated byCRFandCRF-likepeptides,forexample, AL, releaseofPOMC-derivedpeptidesfromtheIL is 1983). Similartotheregulationofcorticotrophsin al. for example,GABA,alsoparticipate(Tomiko et ( and subsequentregulationofCRFreceptorexpression peptides Biosynthesis ofPOMC-derived e al. 1981). Thishighbasalrateof secretion Tilders et Beaulieu et al. 1984),althoughotherneurotransmitters, Published byBioscientifica Ltd. 2 cretagogues suchasacetylcholine,serotonin,and receptorantagonist),suggestingsignalingthrough Early on,dopaminewasshowntoeffectivelyinhibit Further studiesinperifusedratILdemonstrated Downloaded fromBioscientifica.com at09/29/202106:18:27PM . 1992). Notably, these twoet al -MSH andβ 56 -endorphin. : 4 2 β receptors -LPH and T90 via freeaccess Journal of Molecular Endocrinology Endocrinology byRogerCone( Anderson ofMolecularpublished inthisspecialissue ofJournal Sharon Wardlaw (Wardlaw 2011)andanotherreview regulation, readersaredirected toarecentreviewby on POMCneuronsinthe arcuate nucleusand their appetite andobesity. Foramorecomprehensive review interesting moleculeinthehomeostaticregulation of Yaswen et al. 1999).Inthisregard,POMCisaparticularly ( as wellACTHdeficiencyandhypopigmentation in humansandmousemodelsleadstosevereobesity, in humanhealth.Indeed,defectivePOMCprocessing demonstrating the importance of this signaling pathway with inheritedhumanobesityin2008(Looset al. 2008), Mutations inMC4Rwerefirstreportedtobeassociated . 2001, energy balance(Cowleyet al in areasofthebrainstemimportantregulating 4 receptors(MC4Rs)inotherhypothalamicareasand food intakethroughα in vivoandareprimarilyknowntocentrallyregulate humeral signals,asindicatorsofperipheralenergystores are responsive to leptin and insulin, and other neural and those fromtheIL:α peptides producedinthehypothalamusaresimilarto see skin andtheplacenta(forreviewreferenceswithin, identified inwhichPOMCisexpressed,forexample,the site forPOMCexpression;othertissueshavealsobeen hypothalamus peptides fromthearcuatenucleusof Regulation ofsecretionPOMC-derived factors. andinhibitory these stimulatory the finaloutcomedependingoncompetingactionof glandinvolvesmanyfactorswith AL andILofthepituitary regulation ofsecretionPOMC-derivedpeptidesfromthe peptides deliveredtothecirculation. Taken together, the peripheral tissuesresultsincontrollingthelevelsofPOMC between signalingmoleculesfromthehypothalamusand however, itcanbeseenfromthissectionthattheinterplay peptides, fromtheIL;toomanytoadequatelydescribehere, secretion ofpeptidehormones,specificallyPOMC-derived α and bromocriptinesignificantlyreducedthesecretionof indicated that8-bromo-cAMPsignificantlyincreased this baseline(Tilders α could stimulatethereleaseofvariouspeptides,including Jackson -MSH (Castro et al.1989). -MSH, ACTH,andβ-endorphin-likepeptidesabove http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0323 Thematic Review Stevens &White2010).Asalreadystated,thefinal There are many studies investigating the regulation of There aremanystudiesinvestigatingtheregulationof et al. 1997, -MSH andβ-endorphin.Theseneurons The arcuate nucleusisthe thirdmajor Krude et al. 1998, t al.1981).StudiesinbovineIL et -MSH actiononthemelanocortin n

cawley ). Lin &Salton2013). Jackson et al. 1999, et al. 2016). andothers Printed inGreatBritain

granules isregulatedbymultiple contributingfactors; depends on calcium. Stimulated release of the mature are releasedinasecretagogue-dependent mannerthat granules arestoredcloseto theplasmamembraneand and themotorproteins,kinesins 2and3.Themature interaction oftheCPEcytoplasmictailwithdynactin microtubule-dependent anterogrademannerthrough The granulescontainingPOMCaretransportedin a enzymes toproducethebioactivepeptidehormones. processed byexopeptidasesandamidatingacetylating of peptidehormoneintermediates,whicharefurther granules mature,POMCiscleavedintoitscomplement two membrane-associatedproteins,CPEandSgIII.Asthe of the RSP at the TGN through interaction with at least prohormone is sorted into the nascent immature granules processing byprohormone-convertingenzymes.The at leastsevenpeptidehormonesdependingonits POMC isamultivalentprohormonecapableofproducing Summary secretion & Masui1982)thatcouldregulatePOMCpeptide multiple receptorstovariousneuropeptides(Shibasaki suggesting that adenomas of this nature may have to modulate the secretion of POMC-derived peptides, Met-enkephalin, andLeu-enkephalinwerealsofound peptides. SubstanceP, thyrotropin-releasingfactor, and oxytocinstimulatedthesecretionofPOMC-derived arginine vasopressin, vasoactive intestinal polypeptide, derived peptides;however, otherneuropeptidessuchas somatostatin-28 suppressedthesecretionofPOMC- Nelson’s syndrome showed that somatostatin-14and adenomafromapatientwithon theculturedpituitary . 2008).Astudy on theproductionofACTH(Billeret al glands, thuslackingthenegativefeedbackofcortisol adenoma thatoccursaftertheremovalofadrenal enlargement ofapreexistingACTH-secreting pituitary is Nelson’s syndrome,whichresultsfromarapid giving rise to Cushing’s disease. Another example adenomamay occurwithanACTH-producingpituitary leading toCushing’s syndrome.Asimilardysregulation to excessiveglucocorticoids(cortisol)incirculation, of glucocorticoids is faulty(Imura 1985). Thisleads excessive levelsofACTHwhenthefeedbackinhibition by CRF, vasopressinaswellotherfactorsleadsto occur. Forinstance,positiveregulationofACTHsecretion POMC-derived peptidesisabnormal,adiseasestatecan Secretion ofACTHintumors peptides Biosynthesis ofPOMC-derived Published byBioscientifica Ltd. in vivo. Downloaded fromBioscientifica.com at09/29/202106:18:27PM When secretion of 56 : 4 T91 via freeaccess Journal of Molecular Endocrinology Arnaoutova I, Jackson CL, Al-Awar OS, Donaldson JG & Loh YP 2003 &Loh YP Donaldson JG Al-Awar OS, Jackson CL, Arnaoutova I, 1997Regulationofthe &Saito H Aoki Y, Oiso Y Iwasaki Y, Katahira M, 1996MortynJonesmemoriallecture–1995.Calciumchecks Antoni FA Ghamari-Langroudi M, Cone RD, Anderson EJP, Carrington S, Çakir I, Yiorkas AM, Sosinsky A, Goldstone AP, Zekavati A, Alsters SI, Buxton JL, 1983Postnatalontogeny Watson S & Akil H Khachaturian H, Alessi NE, Akil H, Shiomi H & Matthews J 1985Inductionoftheintermediate &Matthews J Shiomi H Akil H, 1987Corticotropin- &Catt KJ Hauger RL Millan MA, Aguilera G, References (Grant #HD000056). Human Development,NationalInstitutesofHealth,Bethesda,MD,USA the EuniceKennedyShriverNationalInstituteofChildHealthand This workwassupportedbytheIntramuralResearchProgramof Funding perceived asprejudicingtheimpartialityofthisreview. The authorsdeclarethatthereisnoconflictofinterestcouldbe Declaration ofinterest normal anddiseasedstatesinvivo. generation, roles,andregulationofthesepeptidesin investigators toaskmorestringentquestionsaboutthe to thepioneersinthisfield.Thisstrongbaseallowsfuture broad andsubstantialfoundationinknowledge,thanks and representsanareaofphysiology, whichnowhasa and itsbioactivepeptideshasbeenstudiedextensively biosynthesis, sorting, trafficking, and secretion of POMC the hypothalamusrespondtoleptin.Thesubjectof inhibition ofdopamine,whereasPOMCneuronsin by glucocorticoids.Melanotrophsareunderthetonic acting onthecorticotrophs,whichinturnareinhibited primarily amongthemiscorticotrophin-releasingfactor DOI: 10.1530/JME-15-0323 http://jme.endocrinology-journals.org Thematic Review of theCell carboxypeptidase Erequiresinteraction withARF6.MolecularBiology Recycling ofRaft-associatedprohormone sortingreceptor (doi:10.1210/endo.138.5.5121) the commonsecretagogues.Endocrinology rat proopiomelanocortingeneexpressioninAtT-20 cells.I:effectsof (doi:10.1111/j.1365-2826.1996.tb00703.x) corticotrophs. cyclic AMP–corticosteroidfeedbackinadenohypophysial Molecular Endocrinology of regulation offeedingandenergyhomeostasisbyaMSH.Journal 201660YEARSOFPOMC: &Litt M Gillyard T, Gimenez LE e0131417. disability andhypogonadotrophichypogonadism.PLoSONE morbidly obesefemalewithtype2diabetesmellitus,intellectual Truncating homozygousmutationofCarboxypeptidaseE(CPE)ina . 2015 et al vanHaelst MM, Bridges N, Klaber RE, Holder S, Sciences of acetylatedandnon-acetylatedB-endorphininratpituitary. Life beta-endorphin. bystress:synthesisandreleaseofanonopioidform pituitary Sciences pituitary, andperipheraltissues.AnnalsoftheNewYork Academyof releasing factorreceptors:distributionandregulationinbrain, 33 (Supplement1)57–60. 512 48–66.(doi:10.1111/j.1749-6632.1987.tb24950.x) 14 4448–4457.(doi:10.1091/mbc.E02-11-0758) Journal ofNeuroendocrinology Journal Science 56 T157–T174.(doi:10.1530/JME-16-0014) 227 424–426.(doi:10.1126/science.3155575) n

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