Signal Transduction

Question 1

Classification of signal transduction

Answer 1

• can be classified in several ways:
  
  1. physiological system (e.g. Neural)
  2. nature of origin of extracellular signal
  3. type of receptor involves

-examples: contact-dependent, paracrine, synaptic, endocrine

Question 2

Signal transduction simplified

Answer 2

• ligand bind to specific receptors inducing a conformation change
• signals are propagated by transient second messengers
• protein phosphorylation amplifies signalling cascades
• G proteins act as molecular switches
• modular domains enhance signal efficiency and regulation
• Signals can be turned off, or can become desensitized

Question 3

Receptor-ligand binding

Answer 3

• except for membrane permeable signals (steroid, NO), most extracellular signals (ligands) must bind a membrane receptor (integral membrane protein) to initiate signalling
• simple receptors exhibit saturation behaviour with a dissociation constant of Kd=[RxL]/[R][L]
• the result is usually a conformational change to activate the receptor, which then transduces the signal across the membrane

Question 4

Agonists vs antagonists

Answer 4

• an agonist is a ligand that initiates a biological response
• an antagonist binds the receptor but causes no response (similar to a competitive inhibitor)
• adenosine is a natural agonist for adenosine receptor
• caffeine is an antagonist of adenosine receptors (binds but doesn’t elicit a response

Question 5

Second messengers

Answer 5

• kept at low intracellular levels until needed, then are transiently generated from abundant cellular precursors
  1. Cyclic AMP is derived from ATP by activation of adenylate cyclase (eg/ GPCR-coupled adrenergic response) can be removed by phosphodiesterase
  2. Hydrolysis of phospholipids gives rise to a variety of 2nd messengers (diacylglycerol, inositol-3-PO4)
  3. Cystolic free Ca2+ is normally <0.1uM but rises 100-fold from extracellular or organellar sources upon ion channel activation (eg. In skeletal muscle contraction, calmodulin activation, etc.)

Question 6

Protein kinases

Answer 6

• 518 encoded in human genome)
• catalyst the transfer of PO4 2- from ATP to specific substrate proteins (at Ser, Thr, Tyr hydroxyl groups)
• protein kinases have conserved catalytic domains and regulatory domains
• their action is reversed by protein phosphatases

Question 7

Protein phosphorylation

Answer 7

• phosphorylation may modify target activity and/or provide docking sites for additional signalling proteins
• their targets are often also protein kinases, thus amplifying a signalling cascade at each step
• their action is reversed by protein phosphatases
• PK pathways are highly conserved and regulated, but re generally slower than Ca2+ signalling

Question 8

G proteins

Answer 8

• act as switches to turn pathways off and on
• when bound to GTP they bind and activate downstream proteins and events
• also act as timers due to their intrinsic GTPase activity, which can be further regulated by other proteins
• 2 major classes:
  
  1. heterotrimeric: (aBY) are directly activated by GPCRs (3 subunits)
  2. monomeric g-proteins: play an important role in many cell processes (1 subunit)

Question 9

2 major receptor types

Answer 9

• G protein coupled receptors (GPCR)

- receptor tyrosine kinase (RTK)

Question 10

GPCRs

Answer 10

• span the bilayers as seven transmembrane a helices
• GPCRs are encoded by about 800 human genes and comprise 50% of know drug targets
• bind a variety of Logan’s in a cleft formed by helices: epinephrine, glucagon, serotonin, angiotensin etc
• one ligand can bind multiple receptors

Question 11

GPCR mechanism

Answer 11

• Logan binding causes a conformational change that activates a heterotrimeric G protein
• subsequent activation of downstream enzymes

Question 12

CAMP activation of protein kinase A

Answer 12

• potential regulation points
  
  • GTPase
  • phosphodiesterase (cAMP)
  • protein phosphatase
  • receptor downregulation
  • receptor internalization

Question 13

GPCR internalization/desensitization

Answer 13

• phosphorylation and binding to B-arrestin blocks GPCR association with G proteins and induces receptor internalization
  
  • can also carry out some signalling roles

Question 14

RTK signalling

Answer 14

• receptor tyrosine kinases (RTKs) respond to ligand binding by phosphorylation, resulting in activation of their internal kinase domain and auto- and cross- phosphorylation of selected Tyr residues
• these P-tyr serve as specific docking sites for other proteins, using modular protein domains to recruit a signaling complex and activate many downstream protein responses, such as Ras, the MAP kinase cascade, and transcription factors
• examples: insulin, epidermal growth factor, neurotrophins

Question 15

Ras molecular view

Answer 15

• involved in many RTK signalling pathways
• is a monomeric G protein that is a molecular switch that can interact with multiple downstream signalling proteins and turn on cell proliferation when bound to GTP
• mutations near the active site impair its GTPase activity and leave it constitutively active (always on)
• Ras mutation are present in about 30% of human cancers