Lecture 7 Chapter 15 Cell signaling: Communication Between Cells and Their Environment

1
Lecture 7Chapter 15 Cell signaling
Communication Between Cells and Their Environment
Overview of intracellular signaling Initiation
and responses
Axiom 7 Study hard and stay focused on your
educational goals. But always have a backup
plan! Remember while chance favors the prepared
mind, you cant always be where the lighting
strikes.
2
What are G-proteins?

• G proteins bind GTP guanosine triphosphate.
  Control and amplify intracellular signaling
  pathways
• Exist in two states 1) bound GTP active
• 2) bound GDP inactive

(hormone, GF, drug)
Examples of GTPase proteins Ras, Cdc-42
Fig. 15.1
3
Jennifer studies G-proteins!

• The J Lo motto A detailed understanding of the
  fundamental processes that govern this GTPase
  cycle will provide a basis for discerning how
  cells relay signals.

G-PROTEINS
4
GTPases and disease.

• Damage to these small GTPase switches can have
  catastrophic consequences for the cell and the
  organism.
• Several small GTPases of the Rac/Rho subfamily
  are direct targets for clostridial cytotoxins.
• Further, Ras proteins are mutated to a
  constitutively-active (GTP-bound) form in
  approximately 20 of human cancers.

5
G-proteins are tightly regulated

• 3 types of accessory proteins that modulate
  cycling of G-proteins between GTP/GDP
• 1. GAPs GTPase-activating proteins. Stimulate
  GTP hydrolysis. Inactivate G-protein. Example of
  a GAP PLCb.
• 2. GEFs Guanine nucleotide-exchange factors
  G-protein-coupled receptors (GPCR). Stimulate
  dissociation of GDP (inactive) from G-protein so
  GTP can bind (active).
• 3. GDIs Guanine nucleotide-dissociation
  inhibitors. Inhibit release of bound GDP
  (maintain G-protein in inactive state).

6
The heterotrimeric G proteins transmit signals
from a variety of cell surface receptors to
enzymes and channels

• Stimulated by receptors
• Act on effectors
• Regulated by nucleotide
• exchange and hydrolysis

7
Fig 15.3 The G Protein Cycle
8
GTP is very small and can diffuse rapidly
throughout the cell

• Diffusion-Mediated Random Walk of Signaling
  Proteins
• Schematic representation of a 4 s long random
  walk of (left) a cytosolic protein, (middle) a
  membrane-bound protein, and (right) a receptor.
  Simulated with MATLAB.

9
GTP binds to GTPases. Hold on!

• Motors on vesicle with G-proteins.
• Interesting thought Some motors has GAP domains
  (Myosin-IX)
• G-proteins may act as motor attachment factors.
  (Rabs)

10
Rabs are members of the small G-protein family

• Rab 6 a GTPase for Kinesin
• Rab 27a A GTPase for myosin-Va
• Defects in Rab27a cause Griscelli syndrome

11
Other G Proteins

• Rho Family of GTPases (convergent pathways)
• Cdc 42 actin-dependent membrane ruffling
• Rac actin-dependent membrane ruffling
• Rho actin-dependent focal contacts (FAK),
  stress fibers
• Ras proliferation

12
Ras Activation and the MAP Kinase Cascade

Tyrosine Kinases, G-Protein coupled receptors
See Fig. 15.2
Raf-1 (Map kinase kinase kinase) MEK (MAP
kinase kinase) ERK (MAP kinase)
Nuclear regulatory proteins Cytoplasmic substrates
13
What does Ras interact with?
Raf
14
G proteins

• G protein structure
• G protein regulation

15
Why do we care about the structure of the
structure of G proteins including the Ga and Gbg
interface?

• The a subunit binds and hydrolyzes GTP
• GTP-a dissociates from Gbg (tightly associated)
• Both subunits (a and bg, then activate their
  respective effectors).
• Following hydrolysis of GTP to GDP, subunits
  reassemble and become inactive
• Ergo contact surface between Ga and Gbg has
  major regulatory importance.

Fig. 15.11
16
The regulation of G proteins.
Fig. 15.12
17
A Ribbon Diagram of the G a,b,g

• The heterotrimer consists of an a subunit that
  binds and hydrolyzes GTP and a pair of proteins,
  b and g, that are tightly associated with each
  other.
• The G a subunit is displayed in light blue, the
  Gb in green, and the Gg in dark blue

18
A Schematic of the Gb Propeller Structure

• The schematic shows the relative placement of the
  four sequential strands in each of the seven
  blades. Also shown are the key WD repeat amino
  acids (see Figure 4). The seven symmetrically
  placed surface Asps in the tight two to three
  residue turn between strands b and c are
  indicated by green cirles on the top surface of G
  . These are not the D of WD. The highly conserved
  aromatics at the lower ends of strands a and c
  are shown by blue circles. The Asp of the
  defining WD, potentially exposed on the
  propeller's wider bottom surface, is indicated by
  a red circle.

Each propeller has 4 b-sheets
19
Take Home Message 1.Cells maintain their
signaling outputs by establishing a balance
between the nucleotide exchange rate and the
hydrolysis rate

• What is the favored bound nucleotide in the
  resting cell? G-GTP or G-GDP?

20
Answer G-GDP

• In the basal state, G alpha releases GDP at a
  slow rate (0.002 s-1) compared to its rate of GTP
  hydrolysis (0.05 s-1 for G). This kinetic
  balance ensures a very low population of
  activated G protein molecules, and maintains the
  cell in a resting state.
• Upon binding to agonist, G protein coupled
  receptors accelerate G alpha subunit GDP/GTP
  exchange.  Receptors work as catalysts, achieving
  rate enhancements of up to 104-fold.
• As receptor-driven nucleotide exchange becomes
  fast relative to hydrolysis, the balance of rates
  in the GTPase cycle changes. The new balance
  increases the population of GTP-bound species,
  thus shifting the cell to an activated state. 

21
Why do we need GAPs?

• To buy kakis

22
Answer

• The kinetic barrier to GTP hydrolysis is
  substantial, allowing G proteins to maintain the
  active signaling state for seconds, potentially
  hours. Hence, GTPase-activating proteins, or
  GAPs, are required to assist G proteins in
  hydrolyzing GTP.

23
Take home message 2
Hydrolysis is the turn-off signal that induces
heterotrimeric G protein a subunits (G a  ) to
disengage their effectors.
Note change in structure of GTP versus GDP bound
G protein
24
Why do we need GEFs?
25
Answer

• The somnolescent state attained after hydrolysis
  should be similarly protracted without
  intervention again, the kinetic barrier to
  product (GDP) release is high, even though GTP is
  in 10-fold molar excess to GDP in the cytosol.
  Replacement of GDP by GTP in the active site of a
  G protein is the turn-on signal that almost
  invariably requires the assistance of a guanine
  nucleotide exchange factor, or GEF.

26
Do you remember everything from yesterdays
lecture?If you say yes, you get a donut!
If not, then you need to pay attention to
the CREB story. Fact CREB cAMP response
element binding protein. Binds to DNA at the
CRE (cAMP Response Element) and activates
transcription.
27
What does CREB do?

• Landmark papers in 1995 demonstrated that
  cAMP-dependent transcription via CREB enhance the
  formation of long-term memory (LTM)

28
Can eating CREB make you smart? NoBut eating
donuts can make you happy, and happiness is good
psychological health!

• Hopefully donuts do not stimulate activation of
  CREB repressor genes!

29
cAMP is generated from ATP by an enzyme adenylyl
cyclase. AC is regulated by G proteins
30
cAMP activates one or more kinases. What are
phosphatases?
31
Activation of cAMP and Protein Kinase A also play
major roles in response of liver to glucagon or
epinephrine.
Figure 15.7
32
Thursday, IP3, Calcium and Receptor tyrosine
kinases

• END

33
Regulation of PIP2 and PIP3 Synthesis
Green arrows denote stimulatory effects blue
arrows denote synthetic pathways red denotes
inhibitory effect. Feedback inhibitory loop (1).
Cross-talk between receptor signaling pathways
(2). Feed-forward loop (3).
34
Plasma Membrane Functions that Require

• . Function    Phosphoinositide     Possible
  Mechanism    
• Membrane Trafficking    
•      
•      
• Endocytosis    
• PIP2    
• Recruitment of AP2 to membrane initiating
  clathrin coating    
•      
• PIP2    
• Uncoating of clathrin-coated vesicles, through
  synaptojanin-1-mediated PIP2 hydrolysis    
• Regulated exocytosis    
• PIP2    
• Recruitment of CAP protein to sites of vesicle
  fusion    
• Membrane/Cytoskeletal Interface    
• Micovilli formation    
• PIP2    
• Activation of ERM proteins    
• Membrane attachment to cytoskeleton