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Molecular Cell Biology 5068 In Class Exam 2 Exam Number: ___________ November 3, 2015 Please print your name: ______________________________________ Instructions: Please write only on these pages, in the spaces allotted and not on the back. Write your number on each page (not your name), so that we can split them up and grade them anonymously. There are a total of 6 pages including this cover page. You may not use any books or notes, and no electronic aids, including calculators. Answer only in the space provided; short, concise answers are preferred and will be rewarded. Please be as neat as possible.
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MCB5068Exam2November3,2015 MercerSection(24pointstotal)1. Ofthethreetightjunction-associatedmarvelproteins(TAMPs),whichfamilyisthemain
determinantofthecharacteristicsoftheparacellularpathway?(1point)2. WhatisthefunctionofthecytosolicPDZdomainofthetightjunction,singlespanprotein:Junctional
AdhesionMolecule(JAM)?(1point)3. Onacellularlevel,deathischaracterizedbytheinabilitytoreplenishdepletedintracellularATP
stores.Anotherhallmarkofcelldeathisanincreaseinintracellularcalcium.Pleaseanswerthefollowing:(3points)
a) Whydoescalciumincreaseincellsduringcelldeath?
b) Whatistheresponseofgapjunctionstotheincreasedintracellularcalcium?
c) Whatpropertyofgapjunctionsmakesthisresponseimportanttomaintainthehealthofcells
connectedtothedeadcell?4. Pleaseoutlinetheexperimentalmethodusedtodeterminethatapicalandbasolateraldirected
vesicleshavedifferentproteincomplements.(4points)
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5. GPIanchoringofproteintolipidraftsmaybeanimportantsortingsignaltosendsomeproteinstothe______________________________surface.(1point)
6. Forthefollowingions,pleaseindicatewhethertheintracellularorextracellularconcentrationis
typicallyhigherinagenericmammaliancell:(3points)Na+K+Cl-
7. Glucosetransporters(GLUT)areexamplesofproteinsinvolvedinfacilitateddiffusion.Briefly
describetheprocessoffacilitateddiffusion.Makesuretotouchonthedirectionoffluxandbindingaffinityoftheproteinonbothsidesofthemembrane.(2points)
8. TheNa,K-ATPaseisanactivetransportproteinthatusestheenergyfromthehydrolysisofATPto
transport_______(#)Naionsextracellularlyand___________(#)Kionsintracellularly.(2points)]9.TheNa/Caexchangerisabidirectionaltransportproteinthatisexpressedincardiacmyocytes
(musclecells)andfunctionstoremovecalciumfromthecytoplasmbyexchangingitwithsodium.CardiacmyocytesalsoexpresstheNa,K-ATPase.Usingyourknowledgeoftherelativeconcentrationsofions(intravs.extracellularly)andtheimportantroleoftheNa,K-ATPaseinmaintainingtheelectrochemicalgradient,pleasestateandprovideabriefexplanationofthechangeinthecalciumionconcentrationthatyouwouldexpectifaNa,K-ATPaseinhibitorlikedigoxinwasusedonacardiacmyocytecellculture.(3points)
10.Trueorfalse.Iffalse,pleasecorrect(4points).
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A. Inthedirectrouteofapical-basolateralsorting,proteinsachievetheirasymmetricdistributionby
selectivestabilizationorretentionatonecellsurface.(1point)B. Inadditiontoregulatingthebindingofbeltdesmosomestotheactinskeletonß-cateninsarealso
transcriptionfactors(1point)C. Desmoplakinisaspotdesmosomeplasmamembraneproteinthatanchorsmicrotubulefilaments.
(1point)D. Generally,theNa,K-ATPaseislocatedexclusivelyonthebasolateralmembrane.(1point)
ColinNicholsSection(21points)1. Theplasmamembraneofcellsisaselectivelypermeablebarrier.Pleasenameonetypeofmolecule
thatisfreelypermeabletothisbarrierandanothertypethatrequiresfacilitationbyaproteincarrierorchannel.(2points)
2. Usingtheconceptofionselectivitythroughsizeorfilter,pleaseexplainwhyNaionsdonotpass
throughionchannelsthatareselectiveforKions.(2points)3. PleasedescribetheNernstpotentialofanion.Iftherestingmembranepotentialismorenegative
thantheNernstpotentialforacation,willthecationmoveintraorextracellularlyinresponsetoanincreaseinconductanceforthation?(3points)
4. ContrasttheNernstequationfromtheGoldman-Hodgkin-Katz(GHK)equation.WhydoestheGHK
equationprovideabetterdescriptionofmembranepotentialasafunctionofpotassiumconcentration?(4points)
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5. Labelwhata,b,c,d,anderepresentonthefigurebelow:(5points)6. BrieflyexplainhowtheK-ATPchannelfunctionsasametabolicsensorofthecell.Besuretomention
thechannels’responsetocellularsubstratesandwhatthepresenceofthesesubstratesindicateintermsofcellularmetabolicstate.(3points)
7. Trueorfalse.Iffalse,pleasecorrect(2points). A.Biologicmembranesfunctionascapacitorswithcapacitanceofthemembranebeinginversely
proportionaltoareaanddirectlyproportionaltomembranethickness(1point) B.Theshapeofanactionpotentialisdependentonthecomplementofionchannelsexpressedbythe
excitablecellfiringit.(1point)
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RonBoseSection(20Points)1. Howareproteinsseparatedby2Dgelelectrophoresis?Whatisthemajorlimitationofthis
technique?(3points)2. Inmassspectrometry,trypticpeptidesarebrokenintofragmentionsbycollisionwithaninertgas.
Measuringthemassofthesefragmentionsgivesusafinalspectrumthatisdifficulttodistinguish.Thisspectrumcontains:(4points)1)2)3)4)
3. MetaboliclabelingandquantitativeproteomicscanbeusedtomapHer2Tyrosinekinasesignaling.
Describetheexperimentalconditionsthatallowyoutodifferentiatebetweenthetargetproteinscontainingphospho-Tyrosineresidues.Howdotheseconditionsaffectthefinalspectrum?Howistheamountofproteinquantifiedbaseduponthemassspectrometryresults?(3points)
4. Nuclearhormonereceptorsdimerizeandbindtohormoneresponseelements(HREs)toregulate
geneexpression.WhatisthegeneraldifferencebetweenHREsboundbyhomodimersversusthoseboundbyheterodimers?(1point)
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5. Listtwowaysthatproteinkinasescanberegulated.(2points)6. Inclass,severalexamplesofhowmisregulatedsignaltransductionpathwaysconferhumandisease,
werediscussed,including:• Her2/EGFR• β-adrenergicreceptors• Renin-Angiotensinsystem• EPOdeficiency
Choosetwoofthesepathways.
Namethediseaseassociatedwithmisregulationofeachpathway(cancer,heartdisease,kidneydisease,hypertension,pain/painrelief).(2points)
Whattypeofpathwayisimplicatedineachdisease(cytokinereceptor,cAMP/secondmessenger,nuclearhormonereceptor,RTK,GPCR)?(2points)
Nameadrugortreatmentusedtotargetthesepathwaysfortreatment.(2points)7. (True/False)The9;22translocationresultsinachromosomalrearrangementcalledthePhiladelphia
chromosomeandafusionproteincalledBCR-ABLinchronicmyeloidleukemia(CML).ThetreatmentofCMLwasrevolutionizedbythedrugGleevec,amonoclonalantibodythattreatsthediseasebyrecognizingandbindingtothefusionprotein.(1point)
KenBlumerSection(35points)1. Listthefourmodesofcellularcommunication.(4points)
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2. Listthethreebasicmodesbywhichsignalscanbetransmittedwithinacell.(3points)3. Thisgraphrepresentstheresultsofaligandbindingassayinordertodeterminetheamountof
insulin-specificreceptorspresentpercell.Labelthethreecurvesonthegraph,thenbrieflydescribethecalculationorexperimentalconditionsusedtogeneratethecurve.(6points)
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4. Scatchardplotsareusedinordertomeasurereceptorabundanceonacell.Labelthepointonthe
plotthatrepresentsthetotalnumberofreceptorsonacell.Whatdoestheslopeofthelinerepresent?Whatcanyoudeducefromthisgraphiftheslopeofthelineissteeper?(3points)
5. Receptorsareresponsibleforgeneratingsecondmessengersinordertotransmitsignalsintoand
throughoutthecell.Nametwoofthesesecondmessengersthatwerediscussedinclassandoneofthedownstreameffectorsthateachofthesemessengersactivates.(4points)
6. WhichsubunitofheterotrimericG-proteinsdosmallG-proteinsresemble?(1point)
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7. TheArgandGlnresiduesintheGTP-bindingsiteareresponsibleforstabilizingthetransitionstate
forGTPhydrolysistooccurbytheG-alphasubunitoftheheterotrimericGprotein.Wouldamutationinthissitebeconsidereddominant-negativeordominant-positive?Brieflyexplain.(2points)
8. SmallGproteinsareactivatedbyguaninenucleotideexchangefactors(GEFs)thathaveaDbl
homology(DH)domainresponsibleforcatalyzingthenucleotideexchangeofGDPforGTP.ListthreewaysthatthisGEFcanbeactivatedinordertointeractwithitstargetsmallGprotein.(3points)
9. WhatarethefourstepsinvolvedinactivatingreceptorTyrosinekinases(RTKs)anditsdownstream
effectors?(4points)
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10. ProgesteroneisresponsibleforinducinggerminalvesiclebreakdownofXenopusoocytesviatheMAPKkinasecascade.Ifweaddaconcentrationofprogesteronethathalf-maximallyactivatesMAPK,doweseeagradedoraswitch-likeresponseoftheMAPKpathway?Describetheresultsfromthisexperimentthatwouldallowyoutomakethisconclusion,includingwhatyouwouldmeasureinordertoevaluateactivationofthepathway.(3points)
11. Phosphoinositide4-phosphate(PIP2)isphosphorylatedbyPI3K,resultinginPhosphoinositide4,5-
bisphosphate(PIP3).WhatproteincanremovethisphosphategroupinordertoreducePIP3signaling?(1point)
12. (TrueorFalse)Gproteincoupledreceptors(GPCRs)haveaconserved5transmembranedomain
architectureandarerobustswitcheswithmultipleeffectors.(1point)