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.

Exam Number:______

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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)