Mechanisms of Cell Signaling

Question 1

name 4 major classes of receptors

Answer 1

1. ion channels
2. steroid receptors (cytoplasm or nucleus)
3. protein kinase (enzyme linked) receptors
4. 7-transmembrane GCPR

Question 2

what is wrong with the receptor in cystic fibrosis?

Answer 2

recessive disease, LOF mutation in CFTR chloride (Cl-) ion channel

normal ligand of CFTR is ATP

impaired channel function —> abnormal salt transport

—> thick mucus build-up in respiratory epithelial cells

Question 3

how does tetrodotoxin (TTX) cause death?

Answer 3

(pufferfish toxin)

structurally similar to hydrated Na+ but larger -> binds sodium ion channels irreversibly

—> disrupts action potential propagation -> diaphragm paralysis and death

Question 4

major families of steroid hormones (5)

Answer 4

1. androgen/testosterone
2. estrogen/estradiol
3. progesterone
4. glucocorticoid/cortisol
5. mineralcorticoid/aldosterone

Bind receptor in cytosol or nucleus to regulate gene expression

derived from cholesterol

Question 5

describe estrogen signaling

Answer 5

steroid hormone, derived from cholesterol

1. ER attached to chaperone protein in cytosol in absence of ligand
2. estrogen binding induces conformational change in ER - dissociates from chaperone to expose nuclear import signal
3. ER-ligand complex homo-dimerizes
4. dimerized receptors enter nucleus to bind ERE (DNA promoter sequence, estrogen response element)

Question 6

tamoxifen

Answer 6

estrogen receptor antagonist, selective for breast tissue

represses estrogen over-expression in breast cancer

Question 7

Addison’s disease, Cushing’s syndrome, and Kallman syndrome have this in common

Answer 7

steroid-hormone related diseases

Addison’s - autoimmune adrenal insufficiency

Cushing’s - secondary hyperaldosteronism, overproduction of ACTH

Kallman - delayed puberty, developmental disorder

Question 8

describe structure of protein kinase receptors

and what are two subfamilies

Answer 8

extra cellular domain (binds ligand)
trans-membrane domain
cytoplasmic domain (kinase activity)

major types (defined by amino acid phosphorylation targets):
1. tyrosine kinases
2. serine/threonine kinases

Question 9

phosphotases remove phosphate group by what mechanism (organic chemistry)

Answer 9

hydrolysis

Question 10

in protein kinase receptor activation, ligand binding to extracellular domain induces monomer dimerization that brings cytosolic protein kinase domains into proximity with one another and phosphorylate each other.

Cytosolic proteins are then recruited to phosphorylated cytoplasmic domains, which induces signaling cascade via _____

Answer 10

Ras-MAPK pathway
(Ras - small G-protein)

ultimately activates TFs vis phosphorylation in nucleus.

[MAPK is only one downstream protein of activated ras-GTP]

Question 11

describe each of these core elements of the Ras_MAPK signaling pathway (in protein kinase receptor signaling):
a. Grb
b. GEFS
c. SoS
d. Ras
e. G-proteins
f. GAPS

Answer 11

a. Grb (G protein receptor binding): contains SH2 domain that binds tyr-phosphorylated receptor cytoplasmic domain, recruits GEFS (such as SoS)
b. GEFS (guanine nucleotide exchange factors): activate G-proteins by promoting dissociation of GDP for GTP
c. SoS (son of sevenless): GEF that binds Grb and activates small G-proteins such as Ras via GTP/GDP regulation
d. Ras (rat sarcoma): monomeric small G-protein
e. G-proteins: binds GTP/GDP and act as molecular switches, have weak intrinsic GTPase activity
f. GAPS (GTPase activating proteins): inactive G-proteins by stimulating weak, intrinsic GTPase activity of G-proteins

Question 12

describe the nucleotide exchange reaction that occurs in the Ras-MAPK pathway in protein kinase receptor signaling

Answer 12

1. ras-GTP is active
2. GAP binds ras-GTP to inactive
3. ras-GDP is inactive
4. SoS (ras-GEF) binds ras-GDP to activate

(it’s a cycle, Ras is G-protein molecular switch)

Question 13

what do these families of small g-proteins generally do?
a. ras
b. rab
c. ran
d. rac

Answer 13

a. ras family: receptor signaling, cell division (s = signaling)
b. rab family: traffic of membrane vesicles (b = bus)
c. ran family: nucleus/ cytoplasm traffic (n = nucleus)
d. rac family: actin cytoskeleton (c = cytoskelton)

Question 14

How does ras mutation play a role in cancer

Answer 14

ras: small G-protein in ras-MAPK pathway

oncogenic mutations in Ras common in tumors - makes it resistant to GAPs

mutated Ras is constitutively active

Question 15

cause of neurofibromatosis type 1 (in cell signaling)

Answer 15

mutation in NF1, which encodes Ras-GAP (neurofirbomin-1)

mutation in GAP genes results in overactive Ras

Question 16

cell signaling mutation that causes Noonan syndrome

Answer 16

mutation in PTPN11 gene, encodes protein tyrosine phosphatase (SHP2)

results in GOF, hyperactive Ras (defective Ras GAP activity)

[ras-MAPK is pathway downstream of protein kinase receptors]

Question 17

most abundant receptor class in human genome

Answer 17

7-transmembrane receptors (seven alpha helices)

large extra cellular binding domains

most receptor-target drugs are directed at 7-TMRs

roles in vision (Opsins such as rhodopsin), smell (olfactory receptors), mood (serotonin, dopamine, GABA, glutamate), ANS

Question 18

serotonin, dopamine, GABA, and glutamate all have this in common regarding their receptors

Answer 18

receptors are all 7-TMRs

Question 19

what are the components of the 7-TMR trimeric complex?

Answer 19

7-TMRs coupled to large G-protein trimeric complex in cytoplasm (alpha, beta, gamma)

alpha subunit contains ras-like domain regulated by GTP/GDP

alpha and beta/gamma subunits dissociate form each other and receptor cytosolic domain following activation

Question 20

7-TMR signaling mechanism steps

Answer 20

1. ligand binding causes GDP->GTP by receptor cytoplasmic tail (acts as GEF)
2. alpha and beta/gamma subunits dissociate from each other and cytosolic domain
3. subtype of G-protein alpha subunit determines downstream affect :
   a. Gs-alpha ACTIVATES adenylate cyclase —>PKA
   b. Gi-alpha INHIBITS adenylate cyclase —>PKA
   c. Gq-alpha ACTIVATES PLC —> PKC

Question 21

what are the three subtypes of G-protein alpha subunit in 7-TMR signaling and what do they do

Answer 21

Gs-alpha: ACTIVATES adenylate cyclase, signaling target is PKA

Gi-alpha: INHIBITS adenylate cyclase, signaling target is PKA (inhibits formation)

Gq-alpha: ACTIVATES phospholipase-Cbeta (PLC), signaling target PKC

Question 22

how does 7-TMR desensitization occur

Answer 22

signaling is terminated even in presence of ligand

1. beta-adrenergic receptor (7-TMR) binds adrenal gland hormone ligands epinephrine and NT norepinephrine
2. ligand bound receptor becomes substrate for beta-adrenergic receptor kinase (BARK)
3. phosphorylated receptor is bound by beta-arrestin —> blocks re-association of cytosolic domain with G-alpha subunit

Question 23

Gs-alpha of 7-TMR upregulates PKA downstream of ligand binding. What are the intermediary steps

Answer 23

Gs-alpha (subtype of G-alpha monomer) activates adenylate cyclase

AC changes ATP—> cAMP

cAMP activates PKA

PKA causes increase in intracelular calcium

Question 24

cytosolic calcium is maintained at a very low concentration by calcium pumps in the ____

Answer 24

calcium pumps in the plasma membrane and SER

PKA induces receptor-mediated calcium influx, following upstream activation by Gs-alpha of 7-TMR

Question 25

Explain steps of Inositol Triphosphate (IP3) and Calcium Signaling Pathway

Answer 25

1. Gq-alpha-GTP activates PLC
2. PLC hydrolyzes PiP2 into iP3 and DAG
3. iP3 triggers calcium release from SER lumen
4. DAG and Ca2+ together activate PKC

Question 26

describe mechanism of PKC activation

Answer 26

1. absence of signaling, C1 and C2 domains of PKC are arranged with psuedosubstrate occupying active site
2. signaling produces DAG and PS (phosphatidylserine) and calcium release is triggered
3. C1 domain binds DAG and exposes active site
4. C2 domain binds PS when calcium is present —> linker region exposed
5. PKC linker is cleaved to release constitutively active kinase subunit

Question 27

how does calmodulin regulate CAMK

Answer 27

calmodulin is calcium-binding protein: when Ca2+ binds, tertiary structure is altered, and calmodulin activates proteins such as CAMK (Ca2+/calmodulin-activated protein kinase)

CAMKs are self-inhibiting proteins (activated by disinhibition) - serine/threonine kinase proteins

1. absence of signaling - inhibitory subunits block kinase (catalytic) domain
2. during signaling - Ca2+/calmodulin binds inhibitory subunit to expose active site

Question 28

how does tamoxifen work to treat Estrogen-sensitive breast cancer (specifically)

Answer 28

antagonizes estrogen signaling by inhibiting, rather than activating, gene at ERE

does not affect localization to the nucleus (effect is after it gets to the nucleus)