Signal Transduction

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					Signal Transduction
                  Cell-cell signalling at a distance:
     Regulation of haploid and diploid specific genes in yeast mating

                                                                     a

•Each haploid cell secretes a mating type pheromone eg. Alpha factor
•Each haploid cell expresses a mating pheromone receptor eg. Alpha factor receptor
•When a haploid cell ‘receives’ a signal from the opposite mating type, a signal transduction
cascade is initiated
•The consequences of this include arrest in the cell cycle in G1 (so that both cells are 1N),
formation of ‘shmoo’ morphology, and expression of proteins involved in cell fusion
Modes of cell-cell signaling

1.   Direct cell-cell or cell-matrix (integrins and cadherins)

2.   Indirect: Secreted molecules.

A. Endocrine signaling. The signaling molecules are hormones secreted
by endocrine cells and carried through the circulation system to act on
target cells at distant body sites.

B. Paracrine signaling. The signaling molecules released by one cell
act on neighboring target cells (neurotransmitters).

C. Autocrine signaling. Cells respond to signaling molecules that they
themselves produce (response of the immune system to foreign antigens,
and cancer cells).
                  Cell Membrane
                  Relays (‘transducers’) and
                     second messengers

Signal molecule                    Cell
Steroid hormones

This class of molecules diffuse across the plasma membrane and bind to
Receptors in the cytoplasm or nucleus. They are all synthesized from cholesterol.

They include sex steroids (estrogen, progesterone, testosterone)
             corticosteroids (glucocorticoids and mineralcorticoids)

Thyroid hormone, vitamin D3, and retinoic acid have different structure and
function but share the same mechanism of action with the steroids.

Steroid Receptor Superfamily. They are transcription factors that function either
as activators or repressors of transcription.

Steroid Hormone Biosynthesis P450 superfamily
Cholesterol Pregnenolone

                              threonine (Thr)
       serine (Ser)
         +                   H3N+   C     COO
      H 3N   C     COO
                                     CH OH

Many enzymes are regulated by covalent attachment
of phosphate, in ester linkage, to the side-chain
hydroxyl group of a particular amino acid residue
(serine, threonine, or tyrosine).
                     Protein Kinase             O

  Protein        OH + ATP      Protein      O   P    O + ADP
            Pi                        H2O
                   Protein Phosphatase

 A protein kinase transfers the terminal phosphate of
  ATP to a hydroxyl group on a protein.
 A protein phosphatase catalyzes removal of the Pi by
Phosphorylation may directly alter activity of an
enzyme, e.g., by promoting a conformational change.
Alternatively, altered activity may result from binding
another protein that specifically recognizes a
phosphorylated domain.
 E.g., 14-3-3 proteins bind to domains that include
  phosphorylated Ser or Thr in the sequence
  RXXX[pS/pT]XP, where X can be different amino
 Binding to 14-3-3 is a mechanism by which some
  proteins (e.g., transcription factors) may be retained in
  the cytosol, & prevented from entering the nucleus.
                     Protein Kinase             O

  Protein        OH + ATP      Protein      O   P    O + ADP
            Pi                        H2O
                   Protein Phosphatase

Protein kinases and phosphatases are themselves
regulated by complex signal cascades. For example:
 Some protein kinases are activated by Ca++-
 Protein Kinase A is activated by cyclic-AMP
Adenylate Cyclase (Adenylyl
                                   cAMP              NH2
Cyclase) catalyzes:
  ATP  cAMP + PPi                                               N
Binding of certain hormones
(e.g., epinephrine) to the outer                     N           N
surface of a cell activates
                                          H2             O
Adenylate Cyclase to form              5' C 4'
                                                 H           H    1'
cAMP within the cell.              O
                                           H 3'              2' H
Cyclic AMP is thus considered            P  O                OH
to be a second messenger.                  O-
Phosphodiesterase enzymes                         NH2
  cAMP + H2O  AMP                       N

The phosphodiesterase that                                    N
cleaves cAMP is activated by
phosphorylation catalyzed by           H2             O
                                    5' C 4'
Protein Kinase A.                             H           H    1'
                                        H 3'              2' H
Thus cAMP stimulates its own          P  O                OH
degradation, leading to rapid   O
turnoff of a cAMP signal.
Protein Kinase A (cAMP-Dependent Protein Kinase)
transfers Pi from ATP to OH of a Ser or Thr in a
particular 5-amino acid sequence.
Protein Kinase A in the resting state is a complex of:
   • 2 catalytic subunits (C)
   • 2 regulatory subunits (R).
Each regulatory subunit (R) of Protein Kinase A
contains a pseudosubstrate sequence, like the
substrate domain of a target protein but with Ala
substituting for the Ser/Thr.
The pseudosubstrate domain of (R), which lacks a
hydroxyl that can be phosphorylated, binds to the
active site of (C), blocking its activity.
R2C2 + 4 cAMP  R2cAMP4 + 2 C
When each (R) binds 2 cAMP, a conformational
change causes (R) to release (C).
The catalytic subunits can then catalyze
phosphorylation of Ser or Thr on target proteins.

PKIs, Protein Kinase Inhibitors, modulate activity of
the catalytic subunits (C).
  kinase cascade from
   extracellular mitogen to
   transcription control in nucleus
Epidermal growth factor &
growth factor receptor bound
   protein 2
guanine nucleotide exchange
   factor SOS
G Protein Signal Cascade
Most signal molecules targeted to a cell bind at the cell
surface to receptors embedded in the plasma membrane.
Only signal molecules able to cross
the plasma membrane (e.g., steroid
hormones) interact with intracellular
A large family of cell surface
receptors have a common structural
motif, 7 transmembrane -helices.
Rhodopsin was the 1st member of
this family to have its 7-helix
structure confirmed by X-ray
crystallography.                        Rhodopsin   PDB 1F88
 Rhodopsin is unique in that it
  senses light.
 Most 7-helix receptors have
  domains facing the extracellular
  side of the plasma membrane that
  recognize & bind particular
  signal molecules (ligands).
                                          Rhodopsin   PDB 1F88
The signal is passed from a 7-helix
receptor to an intracellular G-protein.
 Seven-helix receptors are thus called GPCR, or
  G-Protein-Coupled Receptors.
 Approx. 800 different GPCRs are encoded in the
  human genome.
G-protein-Coupled Receptors may dimerize or form
oligomeric complexes within the membrane.
Ligand binding may promote oligomerization, which
may in turn affect activity of the receptor.
Various GPCR-interacting proteins (GIPs) modulate
receptor function. Effects of GIPs may include:
 altered ligand affinity
 receptor dimerization or oligomerization
 control of receptor localization, including transfer to
  or removal from the plasma membrane
 promoting close association with other signal proteins
 G-proteins are heterotrimeric, with 3 subunits , b, g.
 A G-protein that activates cyclic-AMP formation
  within a cell is called a stimulatory G-protein,
  designated Gs with alpha subunit Gs.
 Gs is activated, e.g., by receptors for the hormones
  epinephrine and glucagon.
  The b-adrenergic receptor is the GPCR for


                    GPCR                                                      plasma
The  subunit of                                                             membrane

a G-protein (G)        gg    cytosol
binds GTP, &                                                      AC
                        GDP bbGTP
can hydrolyze it
to GDP + Pi.         GTP      GDP                             ATP cAMP + PPi

 & g subunits have covalently attached lipid anchors that
bind a G-protein to the plasma membrane cytosolic surface.
Adenylate Cyclase (AC) is a transmembrane protein, with
cytosolic domains forming the catalytic site.


                 GPCR                                                      plasma
The complex                                                               membrane

of b & g
                     gg    cytosol
subunits Gb,g                                                  AC
                     GDP bbGTP
inhibits G.
                  GTP      GDP                             ATP cAMP + PPi

The sequence of events by which a hormone activates
cAMP signaling:
1. Initially G has bound GDP, and ,b, & g subunits
are complexed together.


         GPCR                                                      plasma

             gg    cytosol
             GDP bbGTP

          GTP      GDP                             ATP cAMP + PPi

2. Hormone binding to a 7-helix receptor (GPCR)
causes a conformational change in the receptor that is
transmitted to the G protein.
The nucleotide-binding site on G becomes more
accessible to the cytosol, where [GTP] > [GDP].
G releases GDP & binds GTP (GDP-GTP exchange).


        GPCR                                                      plasma

            gg    cytosol
            GDP bbGTP

          GTP     GDP                             ATP cAMP + PPi

3. Substitution of GTP for GDP causes another
conformational change in G.
G-GTP dissociates from the inhibitory bg complex &
can now bind to and activate Adenylate Cyclase.


         GPCR                                                      plasma

             gg    cytosol
             GDP bbGTP

          GTP      GDP                             ATP cAMP + PPi

4. Adenylate Cyclase, activated by the stimulatory
G-GTP, catalyzes synthesis of cAMP.
5. Protein Kinase A (cAMP Dependent Protein Kinase)
catalyzes phosphorylation of various cellular proteins,
altering their activity.
Turn off of the signal:
1. G hydrolyzes GTP to GDP + Pi. (GTPase).
The presence of GDP on G causes it to rebind to the
inhibitory bg complex.
Adenylate Cyclase is no longer activated.
2. Phosphodiesterase catalyzes hydrolysis of
   cAMP  AMP.
Turn off of the signal (cont.):
3. Receptor desensitization occurs. This process varies
with the hormone.
 Some receptors are phosphorylated via specific
  receptor kinases.
 The phosphorylated receptor may then bind to a
  protein b-arrestin, that promotes removal of the
  receptor from the membrane by clathrin-mediated
4. Protein Phosphatase catalyzes removal by
hydrolysis of phosphates that were attached to proteins
via Protein Kinase A.
Signal amplification is an important feature of signal

 One hormone molecule can lead to formation of
  many cAMP molecules.

 Each catalytic subunit of Protein Kinase A
  catalyzes phosphorylation of many proteins during
  the life-time of the cAMP.
 The stimulatory Gs, when it binds GTP,
  activates Adenylate cyclase.
 An inhibitory Gi, when it binds GTP, inhibits
  Adenylate cyclase.
Different effectors & their receptors induce Gi to
exchange GDP for GTP than those that activate Gs.
In some cells, the complex of Gb,g that is released
when G binds GTP is itself an effector that binds to
and activates other proteins.
Structure of G proteins:                          PDB 1GIA

The nucleotide binding site
in G consists of loops that
extend out from the edge of
a 6-stranded b-sheet.
Three switch domains have
been identified, that change GTPgS
position when GTP
substitutes for GDP on G. Inhibitory G
These domains include residues adjacent to the terminal
phosphate of GTP and/or the Mg++ associated with the
two terminal phosphates.

                 GTP hydrolysis                              N

 H   O           O        O        O                         N   N        NH2
             O   P    O   P    O   P    O   CH2
                 O       O       O           H        H
                                            H              H
                                                OH       OH

GTP hydrolysis occurs by nucleophilic attack of a
water molecule on the terminal phosphate of GTP.
Switch domain II of G includes a conserved
glutamine residue that helps to position the attacking
water molecule adjacent to GTP at the active site.
                                PDB 1GP2

                                                                           PDB 1GP2

                                           Gb - side view of b-propeller

Gb – face view of b-propeller

 The b subunit of the heterotrimeric G Protein has a
 b-propeller structure, formed from multiple repeats of a
 sequence called the WD-repeat.
 The b-propeller provides a stable structural support for
 residues that bind G.
The family of heterotrimeric G proteins includes also:
  transducin, involved in sensing of light in the retina.
  G-proteins involved in odorant sensing in olfactory

There is a larger family of small GTP-binding switch
proteins, related to G.
Small GTP-binding proteins include (roles indicated):
   initiation & elongation factors (protein synthesis).
   Ras (growth factor signal cascades).
   Rab (vesicle targeting and fusion).
   ARF (forming vesicle coatomer coats).
   Ran (transport of proteins into & out of the nucleus).
   Rho (regulation of actin cytoskeleton)

All GTP-binding proteins differ in conformation
depending on whether GDP or GTP is present at their
nucleotide binding site.
Generally, GTP binding induces the active state.
   Phosphatidylinositol Signal Cascades

                   O       H2 C   O       C    R2

              R1   C   O     CH           O

                           H2 C   O       P    O

                                          O        H
                                               1    6
                                      OH                 OH
                                               H    OH
                                      2             H        5
               phosphatidyl-          H                  H
                                               3    4
               inositol                        H    OH

Some hormones activate a signal cascade based on the membrane
lipid phosphatidylinositol.
IP3 may instead be phosphorylated via specific kinases, to IP4, IP5
or IP6. Some of these have signal roles.
E.g., the IP4 inositol-1,3,4,5-tetraphosphate in some cells stimulates
Ca++ entry, perhaps by activating plasma membrane Ca++ channels.
Protein Kinase B (also called Akt) becomes activated
when it is recruited from the cytosol to the plasma
membrane surface by binding to products of PI-3 Kinase,
e.g., PI-3,4,5-P3.
 Other kinases at the cytosolic surface of the plasma
  membrane then catalyze phosphorylation of Protein
  Kinase B, activating it.
 Activated Protein Kinase B catalyzes phosphorylation
  of Ser or Thr residues of many proteins, with diverse
  effects on metabolism, cell growth, and apoptosis.
 Downstream metabolic effects of Protein Kinase B
  include stimulation of glycogen synthesis, stimulation
  of glycolysis, and inhibition of gluconeogenesis.
Signal protein complexes:
Signal cascades are often mediated by large "solid state"
assemblies that may include receptors, effectors, and regulatory
proteins, linked together in part by interactions with specialized
scaffold proteins.
Scaffold proteins often interact also with membrane constituents,
elements of the cytoskeleton, and adaptors mediating recruitment
into clathrin-coated vesicles.
They improve efficiency of signal transfer, facilitate interactions
among different signal pathways, and control localization of signal
proteins within a cell.
AKAPs (A-Kinase Anchoring Proteins) are scaffold proteins with
multiple domains that bind to
  regulatory subunits of Protein Kinase A
  phosphorylated derivatives of phosphatidylinositol
  various other signal proteins, such as:
    • G-protein-coupled receptors (GPCRs)
    • Other kinases such as Protein Kinase C
    • Protein phosphatases
    • Phosphodiesterases
AKAPs localize hormone-initiated signal cascades within a cell, and
coordinate activation of protein kinases as well as rapid turn-off of
such signals.
WAVE (Wiskott-Adlrich related) proteins are scaffolding proteins
which interact with actin and recruit other components, including

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