Signaling 2

Question 1

common cell surface receptors

Answer 1

• gated ion channels-neuronal
• trimeric g protein linked receptors- metabolic function
• receptor tyrosine kinases- common receptors for growth factors and often regulate choices between cell division and differentiation

Question 2

basic phosphorylation signaling mechanism

Answer 2

• protein kinases add phosphates to regulate target proteins
• phosphates can be added to serine, threonine, or tyrosine residues in proteins
• can change conformation, activity, or binding affinity of the protein
• protein phosphatases remove phosphates to reverse the regulation

Question 3

basic g-protein signaling mechanism

Answer 3

• G-proteins are slow GTPases that work as molecular on/off switches
• protein conformation changes when GTP is hydrolyzed
• the GTP bound conformation is ON
• Hryolysis of GTP switches to OFF
• in ON, G proteins bind to other enzymes and modify their activity

Question 4

monomeric G proteins (or small)

Answer 4

-receptor linked (Ras), others are important in other types of cellular processes such as vesicle sorting and fusion (Rab), and control of actin cytoskeleton (Rac and Rho)

Question 5

Trimeric G Proteins

Answer 5

• have alpha, beta, gamma subunits

- all known trimeric G-proteins are associated with cell-surface receptor signalling

Question 6

General properties of trimeric G protein receptors

Answer 6

1. Receptors are seven-pass transmembrane proteins that bind EC ligands
2. Ligand binding stimulates interaction with trimeric G protein and exchange of GTP for GDP on alpha subunit (by changing shape of receptor protein)
3. GTP binding causes dissociation of trimeric complexes into two active complexes, both of which modulate the function of other proteins
4. Activated subunits are linked to membrane but can diffuse laterally to bind to targets
5. binding activates the target. Targets include enzymes and channels. activators can be beta/gamma or alpha/GTP
6. the beta/gamma subunits dissociate from target and rebind with alpha, GTP is hydrolyzed to GDP on alpha to stop the signal

Question 7

Gs family

Answer 7

• stimulate adenylate cyclase
• adenylate cyclase increases cAMP production
• cAMP diffuses and activates PKA, which phosphorylates a specific target aa (Ser or Thr) on its substrate proteins
• virtually all cells have PKA, and its effects are different across different cells
• some cells have cAMP-gated ion channels as well

Question 8

PKA activation

Answer 8

• PKA inactive when regulatory subunits are bound
• each reg subunit has 2 cAMP binding sites
• cAMP binds and the catalytic subunits are released

Question 9

example of Gs linked receptor

Answer 9

1. ligand binds to membrane receptor protein
2. Gs protein binds to receptor and GDP to GTP
3. beta/gamma complex activates adenylate cyclase
4. increase in cAMP
5. activation of PKA
6. PKA phosphorylates (activates) Phosphorylase kinase
7. Phosphorylase kinase phosphorylates (activates) glycogen phosphorylase
8. glycogen to glu-1-P
9. Glycolysis

Question 10

Gene regulation by Gs

Answer 10

• same initial steps
• PKA activates transcription of genes that are regulated by the cAMP response element binding protein (CREB)
• CREB-P* binds to CRE sequences in gene promotors

Question 11

Gi- linked receptor activation

Answer 11

• inhibits adenylate cyclase
• leads to reductions in cAMP
• reverses the effects of Gs

Question 12

Gq linked receptors

Answer 12

1. signal molecule binds to membrane receptor protein
   2.Gq binds- GTP-alpha subunit is activated
2. GTP-alpha subunit activates phospholipase
3. phospholipase cleaves DAG from IP3.
4. IP3 activates release of calcium from ER
5. DAG (plus Ca 2+ sometimes) activates PKC–which has many target proteins including gene regulation..diffuses in cell
   Calcium alone can also activate other kinases

Question 13

Calmodulin

Answer 13

• protein that changes conformation when calcium binds to it (4 binding sites)
• Calmodulin then activates Ser/Thr specific CAM kinases, like myosin light chain

Question 14

Smooth muscle contraction

Answer 14

• Ach binds to Gq linked receptor
• IP3 releases calcium
• calmodulin activates
• MLCK activated- myosin- contraction

Question 15

Phorbol esters

Answer 15

irreversibly bind to and activate PKC.

tumor promotor because they permanently stimulate a pathway in cell division, but not induce tumors themselves

Question 16

other types of G-protein families

Answer 16

-Gt- vision

Go-olfactory

Question 17

Receptor Protein tyrosine kinases

Answer 17

common receptors for growth factors and often regulate choices between cell division and differentiation

Question 18

Activation of receptor protein tyrosine kinases

Answer 18

1. binding of a ligand (dimer, GF) induces receptor dimerization, which results in transphosphorylation
2. When other proteins in the cell bind to the phosphotyrosines, their conformation changes and they pass their signal on (protein domains that bind to the tyrosines are SH2)

Question 19

transphosphorylation

Answer 19

each receptor molecule phosphorylates tyrosines on its partner on the dimer

Question 20

SH2 domains

Answer 20

-domains on proteins that bind to the activated phosphotyrosine

Question 21

Binding to phosphotyrosines

Answer 21

• stimulates Phospholipase Cgamma, which activates IP3 and DAG (Calcium, PKC, Calmodulin)
• SH2 domains of SH2-SH3 adaptor protein bind and activate Ras monomeric G-protein pathway
• Monomeric G proteins activated by CTP exchange factors (GEFs) and turned off by GTPase activating proteins (GAPs)–see picture

Question 22

GTP and Ras

Answer 22

• Adaptor (SH2-SH3) binds to Receptor tyrosine kinase (RTK)
• Ras activating protein (GEF) activates Ras
• Ras activates Map Kinase (3 kinases) cascade, which changes protein activity and gene expression
• MapKKK P MAPKK- P MAPK- growth control via p21 and p27, increases CDKs for passage into cell cycle

Question 23

Tyrosine kinase linked receptors

Answer 23

1. Receptor lacks kinase activity, but ligand binding causes receptor dimerization and activates a separate protein tyrosine kinase associated with the receptor protein. Activation causes kinase trans-phosphorylation and receptor phosphorylation. Other proteins with SH2 domains bind to the phosphotyrosines SEE PICTURE
2. family is large and known to cause tumors if mutated

Question 24

example of tyrosine kinase linked receptors

Answer 24

JAK-STAT

• cytokine binds to receptors
• receptor stimulates JAK kinase, which then trans-phosphorylates itself, the receptor and the STAT regulatory protein
• increase in transcription

Question 25

LOOK AT SUMMARY SLIDE

Answer 25

really important