Advanced Cell Bio Exam 2

Front 1

Basic GTPase family

Back 1

GEF exchanges GDP for GTP

GAP accelerate hydrolysis of GTP

Front 2

Sketch GTPase in two conformations

Back 2

p679

Front 3

Roles of 2˚ messengers

Back 3

diffuse across cell rapidly
amplifies signal
integrate signals

Front 4

Sketch figure to explain binding assay for Kd and # of receptors

Back 4

15-10 p682

-Calculate total binding with radioactively labeled ligand and a cell line w/ recepetor
-Calc nonspecific binding w/ cell line lacking receptor.
-Subtract nonspecific from total to get specific binding curve
-Kd is when 50% bind

Front 5

Measuring Kd of low affinity ligands

Back 5

Vary the [ligand] w/in a steady concentration of a competitive inhibitor

Front 6

Graph the effect of triphosphates on glucagon driven cAMP production

-Implications?

Back 6

Most with glucagon and GTP. Other nucleotides actually diminish glucagon's effect. p720

Lead to discovery of G proteins and their relation to GPCR

Front 7

GPCR structure

Back 7

7 transmembrane helices with 22aa each

4 external and 4 internal loops - determine what it signals and recieves

Front 8

Sketch transduction from GPCR ligand binding to active effector

Back 8

p689 - 15-17

-Note FRET is useful in this analysis

Front 9

Sketch activation of rhodopsin

Back 9

p696: R -> transducin -> PDE -> GDP
All in ~50ms with 50,000x amplification

Dark = high cGMP and more neurotransmission

GAP for transuducin Galpha is Gbeta5 and RGS9

Front 10

Rhodopsin kinase

Back 10

Key for shutting off Rhodopsin* when exposed to continued light.

Arrestin binds to fully phosphorylated R* and shuts it down

Front 11

Dark and light adapting rod cells

Back 11

[] of transducin and arrestin change in cytosol and discs

Front 12

Effects of arrestin

Back 12

Recruits other proteins (serves as an adaptor)
1)Promote endocytosis: clathrin
2)MAP kinase cascade: cSrc
3)Activation of c-Jun kinase

Front 13

Dual roles of G proteins on cAMP

Back 13

Glucagon, epinephrine, and ACTH lead to stimulatory Galpha. PGE1 and Adenosine lead to inhibitory Galpha via different GPCR

Front 14

Sketch PKA

Back 14

p701

Front 15

Separation of same second messenger's different signals

Back 15

1)Spatial separation
2)Temporal Control
3)Coincidence detectors in pathway

Front 16

Sketch two basic GPCR pathways w/ their second messengers

1)To PKA
2)To PKC

Back 16

21 march notes

PKA (cAMP)
PKC (IP3) on p710

Front 17

Effects of increased cAMP (PKA)

Back 17

-PP off
-Glycogen breakdown
-Glycogen synthase off

p703

Front 18

Effects of decr cAMP (Phosphoprotein phosphatase)

Back 18

p703

GP off
glycogen synthesis

Front 19

Known hormone inducing cAMP and cellular response in: adipose, liver, ovary, adrenal, cardiac, thyroid, bone, skeletal, intestine, kidney, platlet

Back 19

Table 15-2

p704

Front 20

Long term effects of PKA

Back 20

Transcriptional changes
-CREB binds to cAMP Response elements (CRE) in DNA
-leaves a memory trace

Front 21

Hormone and response to Ca2+ in: pancreas, parotid, smooth muscle, liver, platlet, mast, and fibroblast

Back 21

Table 15-3

p708

Front 22

Phosphatidylinositol to PIP3 protiens

Back 22

PI-4 kinase
PIP-5 kinase
Phospholipase C (the effector)

Front 23

A kinase associated protein (AKAP)

Back 23

Anchors PDE and PKA together on membranes, so that they are spatially associated and can quickly change the signal of cAMP

Front 24

STIM1

Back 24

casuses ER to migrate to plasma membrane and trigger Ca2+ influx if ER Ca2+ lvls are low

Front 25

Sketch Ca2+ / NO pathway

Back 25

p712

-include: PKG, NO synthase, ACH GPCR, Phophoplipase C, and Calmodulin

Front 26

Sketch glycognolysis signal integration (cAMP and Ca2+) in muscle and liver

muscle: neural and hormonal
liver: Hormonal

Back 26

p712

Front 27

glycogen phosphorylase kinase structure

Back 27

alpha beta gamma and delta subunits
g: the catalytic enzyme
aB: are phosphorylated regulatory
d: homologous to calmodulin

Front 28

Common cell surface receptors that modulate gene expression

Back 28

1)Receptor associated kinase: JAK-STAT, TGF-beta
2)Cytosolic kinase: GPCR
3)Protein subunit dissociation Wnt, Hedghog
4)Protein cleavage: Notch/Delta

Front 29

PTK (protein tyrosine kinase) pathways to affect gene expression

Back 29

1)STAT
2)GRB2 -> Ras -> MAP kinase
3)Phos. C -> Ca2+
4)PI3 kinase -> PKB

*3 and 4 also modify other cellular proteins

Front 30

RTK structure and function

Back 30

Two transmembrane proteins dimerize when ligands bind, activation of internal lip, and further phosphorylation

-often asymmetric dimers
-Km of kinase domain for ATP decr

Front 31

Down regulation of RTK's (HER that binds epidermal growth factor, EGF, as example)

Back 31

1)Receptor mediated endocytosis (either internalization or % to lysosome) - Her1
2)monoubiquitination by C

Front 32

Sketch and function of Epo receptors (EpoR)

Also their regulation

Back 32

like a RTK, except dimer binds one ligand and associated JAK kinase becomes phosphorylated

-Short term: Phosphotyrosine phosphatase
-long term: SOCS leading to ubiquitination and degredation

Front 33

Key adaptor scaffold domains

Back 33

(src homology 2) SH2 or (phospotyrosine binding) PTB

Front 34

Sketch activation of STAT

Back 34

p732

Does it include: SH2 domain on STAT

Front 35

Diagram activation of Ras by RTK and MAP kinase pathway

Back 35

p737 and 739

Front 36

Methods used in yeast to solve mating pathways

Back 36

1)Screen sterile mutants for mutations
2)IP of known proteins to find friends (bind to monoclonal antibodies on beads)

Front 37

Sketch IP3 phosphorylation pathway

Back 37

p747

PKB, PDK1, PKD2

Front 38

Blood sugar hormones

Back 38

Incr use (and uptake): epinephrine and cortisol
Incr release: glucagon
Incr uptake: insulin

Front 39

Describe an experiment for translocation

Back 39

p766

Front 40

Roles of cytoskeleton elements

Back 40

Actin-periphery, cell shape, linkages, crawling, myosin associated cytokinesis, muscles.

Microtubules-Tubes prevent crushing. Highways with kinesin & dynein. Spindle fibers.

Intermediate filaments- networks that are strong and link with ECM attachement sites

Front 41

9 roles of actin

Back 41

Epithelia: Microvilli, cell cortex, adherens belt, Mobile Cell: filopodia, lamellipodia, stress fibers,
Functions: moving vesicles, contractile ring, phagocytosis

Front 42

Physical characteristics of actin

Back 42

G=globular, F= filament

36nm in one twist

F-actin grows faster at + end (.12uM vs .60uM Kd)

A three G-actin nucleus skips the lag time

Front 43

Experimentally determining direction of actin growth

Back 43

myosin S1 decoration - arrows point to - end

Front 44

Actin Binding protiens

Back 44

Play regulatory role
1)Profilin: Exchanges ADP for ATP in G-actin
2)Cofilin: Destabilizes ADP-F-actin of - end
3)Thymosin: sequesters G-actin
4)CapZ: caps + end, t4 shrink
5)Tropomodulin: cap - end, t4 grow
6)Formin FH2 domain: accelerates growth through rocking of ring
7)Arp 2/3: nucleates branches
8)NPF: connects Arp to new G-actin

Front 45

Sketch formin

Back 45

p785

FH2 domain interacts with actin
FH1 recruits profilin
RBD activates formin when binds to Rho GTPase

T4 Rho leads to filopodia

Front 46

Sketch WASp

Back 46

p786

RBD binding to Cdc42 (A Rho family GTPase) activates
ACW domain is Nucleation promoting factor (NPF) that recruits G-actin
--A interacts with Arp
--W interacts with actin

T4 Cdc42 leads to lamellipodia

Front 47

Recruitment of actin

Back 47

1)Endocytosis assembly factors = endocytosis
2)Fc receptors = phagocytosis
3)Listeria ActA = bacteria intracellular movement

Front 48

Toxins that block normal actin funtion

Back 48

Cytochalasin D (fungal alkaloid) blocks G-actin addition to (+) end

Latrunculin (sponge toxin) sequesters G-actin.

Jasplakinolide (sponge toxin) stabilizes dimers so nucleates new filaments

Phalloidin (Amanita phalloides) stabilizes filaments by locking subunits together.

Front 49

5 actin cross linking proteins

Back 49

Fimbrin: short parallel gaps - microvilli, filopodia

alpha-actinin: muscle Z line, stress fibers, filopodia

spectrin: network of actin in cortex, Ca2+ inhibits

Filamin: stabilize networks like a spring, leading edge

dystrophin: links actin to membrane

Front 50

Ankyrin

Back 50

Binds spectrin to band 3 membrane protein

Front 51

Key myosin classes

Back 51

I: Moves membrane relative to actin
II: Classic non-polar dimer, symmetric heavy chain head w/ coiled coil tail with light chan "collars"
V: long necked dimer for vesicle transport

Front 52

Diagram 5 steps of myosin "stepping" cycle

Back 52

p798
1-Binds ATP, releases actin
2-Hydrolysis cocks it
3-Binds
4-Release Pi and power strokes
5-ADP released

Front 53

Optical trapping diagram

Back 53

p799

-measures step size and processivity
(V is processive and II is not)

Front 54

Diagram sarcomere with associated proteins

Back 54

p803

myosin, actin, CapZ, tropomodulin, nebulin, titin, tropomyosin/troponin

Front 55

Regulation of smooth muscle contraction

Back 55

Ca2+ activates MLC kinase, phosphorylation of myosin LC leads to unfolding

Front 56

Cool roles of actin/myosin system

Back 56

1)Cytoplasmic streaming in plants
2)Mitosis contractile ring (I at poles, II at cleavage furrow)
3)Budding yeast

Front 57

What do Cdc42, Rac (front), and Rho (back) result in?

Back 57

filopodia, lamellipodia, and stress fibers respectively

Front 58

Comparison of intermediate and mt chart

Back 58

mt: bind GTP, rigid, assembly at few locations, dynamic, polarized, tracks for kinesins/dyneins,
IF: no nucleotide, tensile strength, assemble on prexisting, low turnover, nonpolar, no motor proteins,

Front 59

Key mt locations

Back 59

cilia
axons/dendrites
mitosis

Front 60

Basic mt structure

Back 60

of alpha beta tubulin dimers with GTP on alpha and GDP on beta

Beta is on the + end

Keep assembling if beta still has GTP

Front 61

Centrosome complex

Back 61

gamma tubulin ring complex (gTuRC) in pericentriolar material nucleates mt

Front 62

Types of mt organizing complexes

Back 62

1) Centrosome
2)MTOC at base of axon
3)basal body at cilia
4)spindle poles during mitosis

Front 63

colchicine

Back 63

a drug that sequester tubulin dimers and promotes catastrophe

Front 64

mt associated proteins (MAP)

Back 64

MAP2 ~40nm spacing in dendrites
Tau ~15nm spacing in axons
EB1 = enhances polymerization of + end
kinesin 13 = promote catastrophe of + end

Front 65

kinesin family

Back 65

1: conventional dimer
2:heterotrimeric

*move to + end
*convergent evolution w/ myosin
*Heads bind ATP

Front 66

sketch dyneins

Back 66

main - direction motor protiens

p839

Front 67

dynactin complex

Back 67

Links dynein to cargo

Contains Arp1 polymer, capZ, and p150

Less well attached to mt than dynein, is pushed along

Front 68

known intermediate filaments

Back 68

1)keratins in epithelia (heterodimers of acidic and basic)
2) Desmins prevent damage in muscles
3)neurofilaments stabilize axons
4)lamins support nuclear envelope

Front 69

plectin

Back 69

key cross linker b/t microtubules and IF

Front 70

All results of Cdc42

Back 70

1)Actin assembly in branching fomr
2)Dynein activation
3)mt +end capture