Lecture 11: Signal Transduction Pathways I

Question 1

Type of 2nd messenger that G-linked-protein receptor generates

Answer 1

• cAMP: formed by ATP by a cyclization reaction that removes two phosphates from ATP and joins the see phosphates to the sugar part of the ATP molecule.
• ATP + adenylate cyclase –> cAMP

Question 2

Which enzyme is responsible for the degradation reaction of cAMP to AMP

Answer 2

• cAMP phosphodiesterase.

- cAMP –> AMP.

Question 3

Define metabolic responses to hormone-induced rise in cAMP in various tissues

Answer 3

Hormones inducing the rise in cAMP:
adrenaline in muscle/liver; breakdown of glycogen (storage form of glucose) to make more glucose, immediate form metabolic fuel,
in adipose (fat storage of cells); breakdown of triglycerols (storage form of fatty acids) to make more fatty acids, immediate form of metabolic fuel.
in the heart; increase in contraction rate, which increases blood supply to the tissues.

Question 4

Adrenaline-mediated rise in cAMP is important in mediating the body’s response

Answer 4

to stress such as fright or heavy exercise, when all tissues have an increased the need for glucose and fatty acids.

Question 5

Adrenaline-induced activation of adenylate cyclase is mediated by

Answer 5

β-adrenergic Gs-protein-linked receptors and heterotrimeric Gs proteins.

Question 6

The Gs-protein-linked receptors consist of:

Answer 6

• extracellular ligand-binding domain
• seven transmembrane α helices
• cytosolic Gs-protein-binding domain

Question 7

The heterotrimeric Gs(s = stimulatory) proteins consist of:

Answer 7

• 3 subunits, designated sα, β, and γ

- the sα subunit binds guanine nucleotides (GDP or GTP), which regulate G protein activity

Question 8

Why are they called heterotrimetric Gs proteins

Answer 8

to distinguish them from other guanine nucleotide-binding proteins, such as the Ras proteins.

Question 9

Step in activation of adenylate cyclase by adrenaline

Answer 9

step 1: binding of adrenaline to β-adrenergic Gs-protein-linked receptor activates the receptor by inducing conformational change in binding domain.

step 2: The activated receptor binds to the sα subunit of the inactive heterotrimeric Gs protein.

step 3: Binding to the receptor induces a conformational change in the sα subunit, exchanging GDP for GTP.

step 4: The activated GTP-bound sα subunit dissociates from β and γ, which remain together.

step 5:

• conformational change in the sα subunit
• then diffuses along the cytosolic surface of the plasma membrane until it binds to adenylate cyclase.

step 6:

• Binding to the GTP-bound sα subunit stimulates adenylate cyclase.
• catalyzing the synthesis of cAMP from ATP.

step 7:

• The activity of the GTP-bound sα subunit is terminated by hydrolysis of bound GTP.
• the inactive sα subunit (now with GDP bound) reassociates with the β and γ complex.
• cycle is ready to restart.

Question 10

In adipose tissues (fat storage cells), the cAMP level can be both

Answer 10

• up-regulated and down-regulated by the action of hormones adrenaline and prostaglandin.
• adenylate cyclase is stimulated and inhibited by different receptor-ligand complexes.
  Adrenaline; cAMP high, adenylase cyclase activity high, β-adrenergic receptor, sα (s = stimulatory) subunit of G-protein, β-γ subunits identical.
  Prostaglandin; cAMP low, adenylase cyclase activity low, α-adrenergic receptor, iα (s = inhibitory) subunit of G-protein, β-γ subunits identical.

Question 11

The diverse effects of cAMP in animal cells are mediated by the action of

Answer 11

cAMP-dependent protein kinase (aka protein kinase A).

Question 12

The inactive form of protein kinase A is a

Answer 12

tetramer, consisting of two regulatory ( R ) and two catalytic ( C ) subunits where each R subunit has site B for binding cAMP.

Question 13

Steps in the regulation of protein kinase A

Answer 13

step 1:

• Binding of cAMP to site B induces a conformational change.
• reveals second site for binding of cAMP (site A).

step 2:

• Binding of cAMP to site A leads to release of the catalytic subunits C.
• C are now enzymatically active and can phosphorylate various protein targets.

Question 14

Define fast and slow responses to adrenaline-induced,

cAMP- and protein kinase A-mediated signalling

Answer 14

Fast: protein kinase A phosphorylates many enzymes, increasing or decreasing their enzymatic activities within seconds.

Slow: protein kinase A phosphorylates some transcriptional activators that stimulate the transcription of cAMP-inducible genes within minutes or hours.

Question 15

Two metabolic functions of cAMP-protein kinase A (fast response)

Answer 15

1- inhibits the synthesis of glycogen (glycogen synthase).

2- activation of degradation of glycogen (glycogen phosphorylase).

Question 16

Describe the degradation of glycogen by cAMP activated protein kinase A

Answer 16

1) catalytic subunit of cAMP activated protein kinase A phosphorylates and activates glycogen phosphorylase kinase.
2) Phosphorylated and activated glycogen phosphorylase kinase phosphorylates and activates glycogen phosphorylase, which catalyzes the breakdown of glycogen to glucose.

*both steps require the hydrolysis of ATP.

Question 17

Describe the inhibition of glycogen synthesis by cAMP activated protein kinase A

Answer 17

Catalytic subunit of cAMP-activated protein kinase A phosphorylates glycogen synthase (inactivated by phosphorylation).

Question 18

Result of inhibition of glycogen synthesis

Answer 18

elevation of cAMP and activation of catalytic subunit of protein kinase A which blocks the synthesis of glycogen while stimulating the degradation of glycogen.

Question 19

By how much is the signal amplified by intracellular signalling cascade of events

Answer 19

• 100 000 000 signal amplification.

- protein kinase A : glycogen phosphorylase kinase : glycogen phosphorylase = 1 : 10 : 240.

Question 20

Steps in the rise in the cytosolic cAMP level activating the transcription of cAMP-inducible target genes (slow response)

Answer 20

step 1:

• cAMP bound to both B and A sites.
• The free catalytic subunit of protein kinase A translocates to the nucleus via NPCs.

step 2: Within the nucleus, the catalytic subunit of protein kinase A phosphorylates and activates CREB protein by hydrolysis of ATP.

step 3: Phosphorylated CREB proteins form a dimer that binds to CRE (cis acting regulatory region).

step 4: A transcriptional co-activator called CBP/300 binds to phosphorylated serine in the CRE-CREB complex.

step 5: CBP/300 links CREB protein to the basal
transcriptional machinery, permitting stimulation of transcription of cAMP inducible genes.

Question 21

All genes regulated by cAMP contain

Answer 21

• a cis acting DNA sequence called cAMP-response element (CRE) which binds the phosphorylated form of a transcriptional factor called CRE-binding CREB protein.