PD Principles: Effect of Drugs on Receptors & Cell Signaling; Types of Receptors

Question 1

Ligand-Gated Ion channels:

Answer 1

Ligand
• Cell impermeable
• nACh, GABA, 5-HT Drugs
• Nicotine, gabapentin, diazepam, ondansetron

Secondary Messengers
• Na+, K+, Cl-, Ca2+ Mechanistic information
• Conformational changes alter residues revealing pore
– i.e. Gate/pore opens

Question 2

What are the steps of ligand-gated ion channel signal transduction?

Answer 2

1) Receptor consists of 5 subunits
• Hetero- or homo- pentamers

2) Ligand binds extracellular active site
• Ex: Acetylcholine (two molecules)
and GABA (one molecule; two sites)

3) Conformational change
• Pore opens
• Less selective for ions than Voltage-gated

4) Ion flux
• Down electrochemical gradient

5) Intracellular [ion] increases
• Eventually membrane depolarizes
• Pore closes

Question 3

Voltage-gated ion channel:

Answer 3

VOLTAGE-GATED ION CHANNELS ARE NON-RECEPTORS

Ligand
• NONE – triggered by change in membrane potential

Drugs
• Lidocaine, verapamil (calcium channel blocker)

Second messengers
• Na+, Ca2+, K+, Cl- other ions Mechanistic information
• Major conformational change forms a channel selective for particular ion(s)

Question 4

What are the steps in Voltage-gated ion channel signal transduction?

Answer 4

1) Membrane potential
Depolarization

2) Conformational change
Pore opens

3) Ion flux
Down electrochemical gradient

4) Intracellular [ion] increases
Membrane repolarizes
Pore closes

Question 5

G-protein Coupled Receptors (GPCR):

Answer 5

Ligand
• Cell impermeable (mACh, prostaglandins)

Drugs
o Atropine, propranolol, albuterol

GPCR subunits and 2nd messenger pathways
• Gαs (stimulatory)
o Activates AC → ↑ cAMP, ↑ Ca2+ channels
• Gαi and Gαo (inhibitory)
o InhibitsAC→↓cAMP,↑K+channels
• Gαq
o Activates phospholipase C (PLC )→ hydrolyzes PIP2 → IP3 + DAG → activate protein kinase C (PKC)
• Gα12/13
o Diverse ion transporter interactions

Question 6

What are the most abundant types of receptors?

Answer 6

G-Protein-Coupled Receptors (GPCR)

• 7 transmembrane α-helix domains
• Ligand binds extracellularly →conformational change
• GTP binding regulatory proteins

Question 7

GPCR Activation Mechanism:

Answer 7

1. Inactive complex
     – 7-TM protein
     – Heterotrimeric g-protein
          • α/β/γ subunits are all bound 
     – GDP-bound to α-subunit

2. Ligand binding
   – Activation of receptor
3. Conformational change
   – Receptor and G-protein
4. GDP dissociates
   – GTP associates with α-subunit
5. Dissociation of α-subunit
   – Binds and regulates effectors
6. Dissociation of βγ-subunit
   – Binds and regulates effectors
7. Ligand unbinding
   • GTPhydrolysis
   – Returns to inactive state

Question 8

GPCR: Effector modulation by α-subunit (a):

Answer 8

1. G-protein α-subunit
   Binds downstream targets

2. Modulates second messenger activity 
     – Adenylyl cyclase (AC)
          • Gαs  ↑ AC activity
          • GαiAC activity
     – Phospholipase C
          • Gαq  ↑ PLCβ activity

Question 9

GPCR: Effector modulation by α-subunit (b):

Answer 9

1. Gαs (stimulatory)
   – Activates AC, Ca2+ channels
2. Gαi and Gαo (inhibitory)
   – Inhibits AC, activates K+ channels
3. Gαq
   – Activates phospholipase C
4. Gα12/13
   – Diverse ion transporter interactions

Question 10

GPCR: Effector modulation by βγ-subunit:

Answer 10

1. G-protein βγ -subunit
   Binds ligand-gated ion channels and downstream targets
2. Modulates second messenger activity
   – Sodium and potassium
   – PI3 kinase pathway

Question 11

GPCR: Inhibition of signaling:

Answer 11

1. G-protein receptor kinase (GRK)
   Receptor phosphorylation
2. Binding of arrestins
   Inhibitory proteins
   G-protein subunits cannot bind the receptor

Question 12

Receptor Tyrosine Kinase (RTK):

Answer 12

Ligand
• Cell impermeable
• Insulin, growth factors (i.e. EGF), hormones, etc.

Drugs
     • RTK Signaling Antagonist:
          – Anticancer: trastuzumab, imatinib
     • RTK Signaling Agonist:
          – Insulin lispro (Humalog®)

Receptor
     • Single transmembrane receptor 
     • Fully activated receptor:
          – PhosphorylatedDimer 
          – Inherent kinase activity
               • Autophosphorylation

Second messengers
• SH2 domain / PTB proteins

Question 13

RTK Activation:

Answer 13

Monomers are inactive

Ligand binding results in dimerization

Conformational change ‘opens’ cytoplasmic kinase domain of receptor; now active
– Intracellular domain posses intrinsic kinase activity (trans-PO4)
– Intracellular kinase domain on receptor dimers autophosphorylate Tyr- residues on C-terminus

Question 14

RTK Activation and Signaling:

Answer 14

• SH2-adapter proteins (2nd messengers) are recruited and bind SH2 binding domain on the receptor
• Phosphorylation or dephosphorylation of downstream substrate activates intracellular signaling pathways

Question 15

Cytokine receptor:

Answer 15

Ligand
• Cell impermeable
• Interleukins, erythropoietin, prolactin

Drugs
• Tocilizumab, epoetin-α

Receptor
• Single transmembrane (dimer)
• Lacks inherent kinase activity

Receptor activation
• Dimerize upon binding
• Ancillary kinase proteins
– necessary for activation

Second messengers (accessory)
• Soluble tyrosine kinases
• JaK/STAT pathway

Question 16

Cytokine receptor:

Answer 16

• Monomers are inactive
• Binding results in dimerization
• Ancillary kinases (Janus-Kinase (JAK)) bind c-terminus of receptor

• Substrate phosphorylation propagates signal
– JAK → STAT-P
– STAT-P translocates to nucleus for gene regulation

Question 17

Intracellular receptor – Type I:

Answer 17

Ligand
• Cell permeable
• Endogenous: Estrogen, testosterone

Drugs
• Androgens,cortisol

Second messengers
• None

Mechanistic information
• Cytoplasmic/nuclear
• Ligand binds receptor before DNA

Question 18

Nuclear receptor – Type I steps:

Answer 18

1) Ligand penetrates cell

2) Binding event
– Release of inhibiting complex – Ex: hsp90

3) Structural change
– Conformational change
– Homodimerization

4) Downstream events
     – DNA binding
     – Transcription regulation
     – Receptor activation
     – Signal continuation

Question 19

Intracellular receptor – Type II:

Answer 19

Ligand
• Cell permeable
• Endogenous: Thyroid hormone

Drugs
• Levothyroxine

Second messengers
• None

Mechanistic information
• Nuclear
• Receptor bound to DNA

Question 20

Nuclear receptor – Type II steps:

Answer 20

1. Receptor able to bind DNA in absence of effector
2. Ligand penetrates cell
3. Binding of effector displaces inhibitory unit
4. Downstream events
   – Transcription regulation