Transduction in Endocrinology
Endocrinology 317/319
Department of Biology
University of Massachusetts Boston
Kenneth L. Campbell
Assembled August 2007
What are transducers?
Transducers are proteins that convert the
information in hormonal signals into chemical
signals understood by cellular machinery.
They change their shape & activity when they
interact directly with protein-hormone complexes.
Usually enzymes or nucleotide binding proteins,
they produce 2nd messengers, or change the
activity of other proteins by covalently modifying
them (adding or removing phosphate, lipid groups,
acetate, or methyl groups), or they interact with
other proteins that do these things.
They begin amplifying the energy content of the
original hormone signals.
What are effectors?
Effectors are the enzymes & other
proteins that convert the transduced
hormonal signal into biochemical
changes that generate the cellular
response to hormone binding.
Usually amplify the signal further &
allow cellular work to be done: cell
motion, growth, division, altered
metabolism, secretion,
depolarization, etc.
Transduction:
The biochemical mechanism(s) that allow the transfer of
information between an occupied hormone-receptor & the
molecules within the cell that result in production of a
cellular response.
Ligand
Receptor
Transducer
Amplifiers
Effector
Change in metabolism
Change in transcription/gene-read out
Change in secondary hormone production
Transduction System Concepts
Features of transduction that both alter protein shape & function
Allosteric changes
Phosphorylation
Membrane Receptors
usually for proteins & charged molecules
rapid response systems, sec-min
Intranuclear Receptors
lipids & hydrophobic hormones
longer term responses, min-days
Transduction Pathways Depend on Receptor Types
Ion Channels
Intracellular/Intranuclear Receptor
Steroids (sex, adrenal, vitamin D, sterols)
Thyronines (tri-iodothyronine)
G-Protein Receptors/Serpentine Receptors
cGMP/NOS
cAMP/PKA/CREB
PLC/PKC/Calcium ion
Cytokine & GH Receptors
JAK/STAT
TyrK
Ras/GAP/MEK/MAPK
RAC/Rho
PI3K
PLC/PKC
Cross-talk allows unique responses in specific tissues &/or at specific times.
Transduction Systems I:
• Translate
information in hormone messages into language that can be
interpreted & acted upon by target cells.
• For proteins, peptides, & hormones with a high ionic charge at neutral pH,
receptors are usually integral membrane proteins in the cell
surface. When hormones bind, the receptors interact with membranebound or intracellular transducer proteins to begin the cascade of events
leading to cellular response.
• Some
membrane receptors, e.g., the acetyl-choline receptor, act as ion
channels that open or close in response to hormone binding & induce
changes via changes of the intracellular ion/charge balance.
• For many lipophilic hormones, e.g., steroids or thyronines, receptors are
intracellular, usually intranuclear, proteins. When their specific ligands
bind, the hormone-receptor complexes undergo conformational changes
that allow them to interact with specific hormone recognition sites (HREs)
in the DNA of the regulatory regions of certain genes.
Transduction paths involve
allosteric changes to
proteins &/or chemical
modifications such as
phosphorylation,
methylation, or acetylation.
These change transducer &
effector molecule charges,
shapes & functions. They
often alter intracellular
location & associations.
The paths may yield rapid
metabolic changes – often
via membrane receptors -or trigger longer term
responses via altered
transcription & translation
of proteins – via
intracellular/nuclear
receptors.
Transduction Systems II:
• Transduction
processes involve allosteric changes in receptor &/or
transducer protein shape. Signaling cascades of membrane-bound
receptors almost always involve protein phosphorylation by kinases;
kinases may be activated initially via generation of secondary messengers
produced by allosteric activation of enzymes like adenylyl or guanylyl
cyclase or via unmasking of kinase activities that are part of the
cytoplasmic portions of the receptor proteins themselves.
• Both
allosteric changes &/or phosphorylation, which changes protein
charge, alter protein shape &/or intercellular location & protein
function. These are exactly the changes that trigger a biochemical &
cellular response.
This may occur without intervention of protein
synthesis & therefore may be very rapid, milliseconds to minutes.
• Opening
or closing of ion channels also yields rapid responses either
directly or via the intervention of phosphorylation cascades with the
associated changes in protein functions.
• Intranuclear
receptor transduction also involves phosphorylation &
allosteric changes. It normally triggers changes in gene transcription &
subsequent protein production & frequently modulates changes during a
longer time course of minutes to days.
How many kinds of
transducers are there?
How many kinds of
transducers are there?
How many kinds of
transducers are there?
How many kinds of
transducers are there?
How Significant Are Transduction Systems?
At least 16.4%, or
1/6 of the genome is
devoted to coding
for proteins that are
part of transduction
systems. If many
transcription factors
& cell adhesion
proteins are
included, the
fraction rises to
~25% -- fully one
quarter of the
genome’s entire
Good coded
site oncontent!
transduction
l
V
http://employees.csbsju.edu/hjakubowski/classes/ch331/signaltrans/olsignalkinases.html
Receptor-binding specificity of VEGF family members & VEGFR-2
signaling pathways
Note that some hormones
may act via multiple receptors
depending on hormonal
concentration & receptor
numbers. Conversely, some
receptors may respond to
several related hormones,
again, depending on hormone
concentration & receptor
numbers. The activated
pathways may trigger similar
or dissimilar events & may act
independently, synergistically,
or antagonistically.
Flexible cellular response!
Hiroyuki Takahashi, Masabumi Shibuya, The vascular endothelial growth factor (VEGF)/VEGF receptor
system and its role under physiological and pathological conditions, Clin. Sci. (2005) 109, 227-241
Clinical Science
www.clinsci.org
Can single cells make or sense
more than one hormone at a time?
Yes, cells can make multiple hormones, even of
differing chemical classes, & they can sense
multiple signals -- & integrate them -- all at once.
Examples:
Ovarian granulosa cells make inhibin (protein),
estradiol (steroid), & androstenedione (steroid)
during the follicular phase of the ovarian cycle. At
the same time they respond to FSH & growth factors
(proteins), estradiol (steroid), & thyroxine (amino
acid derivative), along with other hormones.
Anterior pituitary gonadotropes respond to LHRH
(peptide) & inhibin (protein), estradiol, testosterone,
progesterone, & glucocorticoids (steroids) while
they make both FSH & LH (proteins).
The next two slides present links to extensive descriptions of the
known transduction pathways &/or to details on particular steps in
each transduction path.
Jakubowski – Chapter 9 – Signal Transduction
http://http://employees.csbsju.edu/hjakubowski/classes/ch331/sig
naltrans/olsignalkinases.html
pathFinder
pathFinder is a program which finds signal transduction pathways between first,
second, or nth messengers and targets within the cell. The usefulness of pathFinder
consists in its ability to identify all possible signal transduction pathways connecting
any starting component and target for a given set of possible two-component pathways
in the pathFinder database. At present, there are 60 such two-step pathways in the
pathFinder db. Addition of two-step pathways is ongoing. pathFinder can also identify
all pathways connecting a starting component and a target when one or more
intermediate pathway components are removed (excluded). This allows investigation of
minimal activation sets and predicts effects of inhibitors of specific pathways on target
activation from a given nth intracellular messenger. pathFinder is not quantitative:
partial inhibitions and partial activations are not within the purview of pathFinder.
Combinatorial activations, however, can be analyzed using pathFinder, although these
are limited in the present version. Please send your comments or requests for on-line
tutorials on pathFinder to eidenl@mail.nih.gov.
pathFinder was conceived and constructed as a collaboration between Molecular
Science Institute's Larry Lok and Lee Eiden of the NIMH-IRP, and has been adapted
for Web use by Margaret Dayhoff-Brannigan, NIMH-IRP summer intern.
Link to pathFinder
Link to view CCList
intramural.nimh.nih.gov/lcmr/smn/
Basic Schematic of Major Signaling Pathways
homepages.strath.ac.uk/.../BB329/MCSlect6.html
More Detailed Schematic of Major Membrane Receptor Transduction
Pathways for G-Protein Receptors & for
Receptors Triggering Tyrosine Kinase Activity
http://www.ust.hk/~stn/graphs/multiprotein_signal_transduction.html
Interacting
pathways
including
those involved
in cell-cell or
cell-matrix
interactions.
http://www.benbest.com/health/cancer.html
Another
illustration of
the overlaps
& potential
extent of
intracellular
cross-talk
mediated by
the common
transduction
pathways.
Discussion of Specific
Transduction Pathways
cGMP & Protein Kinase G
Glyceride, Phosphatide & Inositide Nomenclature & Relationships
Sperm binding to an egg triggers a transduction
cascade involving phospholipase C, protein kinase
C & a rise in intracellular Ca++ that acts as a wave
passing across the egg cytoplasm beginning at the
point of binding of the sperm. This can be watched
by first injecting the egg with a calcium-sensitive
fluorescent dye like green dextran. Under a
fluorescence microscope, the calcium-bound dye
appears as a red wave.
http://Whitaker1996Contr
ol of Meiotic Arrest.htm
© 1997 Kenneth L. Campbell
Note that in the small G protein pathways the kinases require accessory
proteins to exchange GTP for GDP or to cleave GTP to GDP, that many
of the proteins interact via sites involving phosphorylated tyrosine
residues, & that there are often multiple steps of kinase action each of
which amplifies the original hormonal signal.
Tyrosine kinase-activated
networks often use the
P~Tyr sites as binding
domains where auxiliary
proteins involved in the
subsequent cascade
events may dock, be
allosterically modified &
undergo functional
alteration. SH2 & SH3, src
homology domains 2 & 3,
are the most common of
the docking sites
generated by tyrosine
SH2 domains are common protein motifs that phosphorylation by
tyrosine kinase.
can be found in many different tyrosinekinase mediated signal transduction
pathways. They bind & recognize specific
peptide sequences containing phoshorylated
tyrosines (see figure).
ccs.chem.ucl.ac.uk/research/sh2.shtml
Schematic of the grb2, sos, ras, raf, mek,
mapK, myc, jun/fos (AP-1) pathway leading
from an extracellular growth factor signal
to a change in gene expression.
Zbigniew Walaszek, Margaret Hanausek, Thomas J. Slaga, Combined natural source
inhibitors in skin cancer prevention, Cellscience Reviews 1(3), ISSN 1742-8130.
Many of the transducer proteins are found in multiple forms each of
which is specific to a particular tissue or cellular function.
3D Structures of GTPases in Action:
http://www.cs.stedwards.edu/chem/Chemistry/CHEM43/CHEM43/GTP/Index.htm
Phosphoinositide-3 Kinase (PI3K) plays an important role in paths
associated with cell fate.
http://www.benbest.com/health/cancer.html
Transcriptional Mechanism of Steroids
http://www.aw-bc.com/mathews/ch23/fi23p16.gif
© 2000 Kenneth L. Campbell
Mechanism of T3
4 functional intranuclear T3 receptors: α1, β1,2,3; & 1
nonfunctional receptor, α2. Expression varies with
tissue & developmental stage.
http://www.addison.ac.uk/endocrine_modules/module1/lecturers_
material/html_files/END1.08/index.htm
Cross your eyes, relax, & see if you can see how 2
molecules of steroid receptor, green & yellow,
interact with a specific sequence, SRE, in DNA.
Receptors for steroids, T3, retinoids, vitamin D, &
aryl hydrocarbons all work this way.
Steroid Receptors Bind HREs as
Homo- or Heterodimers
http://gibk26.bse.kyutech.ac.jp/jouhou/image/dna-protein/all/small_S1glu.gif
Receptor Binding to HREs Can Bend
DNA & Alter Transcription
http://gibk26.bse.kyutech.ac.jp/jouhou/image/dna-protein/all/small_S1run.gif
Biochemistry of Metabolism
Signal Transduction
Copyright © 1999-2006 by Joyce J. Diwan.
All rights reserved.
The following slides can be found and downloaded at:
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/signals.htm
serine (Ser)
threonine (Thr)
H
H
H3N+
C
COO
H3N+
C
COO
CH2
CH OH
OH
CH3
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).
O
Protein Kinase
OH + ATP
Protein
Protein
O
P
O + ADP
O
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 hydrolysis.
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
acids.
 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.
O
Protein Kinase
OH + ATP
Protein
Protein
O
P
O + ADP
O
Pi
H2O
Protein Phosphatase
Protein kinases and phosphatases are
themselves regulated by complex signal
cascades. For example:
 Some protein kinases are activated by Ca++calmodulin.
 Protein Kinase A is activated by cyclic-AMP
(cAMP).
Adenylate Cyclase (Adenylyl
Cyclase) catalyzes:
ATP  cAMP + PPi
Binding of certain hormones
(e.g., epinephrine) to the
outer surface of a cell
activates Adenylate Cyclase
to form cAMP within the cell.
Cyclic AMP is thus
considered to be a second
messenger.
NH2
cAMP
N
N
N
N
H2
5' C 4'
O
O
O
H
H 3'
O
P
O-
H
1'
2' H
OH
Phosphodiesterase enzymes
catalyze:
cAMP + H2O  AMP
N
N
The phosphodiesterase that
cleaves cAMP is activated by
phosphorylation catalyzed by
Protein Kinase A.
N
N
H2
5' C 4'
O
Thus cAMP stimulates its
own degradation, leading to
rapid turnoff of a cAMP signal.
NH2
cAMP
O
O
H
H 3'
P
O
O-
H
1'
2' H
OH
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).
R2C2
R2C2
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).
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 receptors.
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
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.
 Approximately 800 different GPCRs are
encoded in the human genome.
PDB 1F88
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
, , .
 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 -adrenergic receptor is the GPCR for
epinephrine.
hormone
signal
The  subunit of
a G-protein (G)
binds GTP, &
can hydrolyze it
to GDP + Pi.
outside
GPCR
plasma
membrane

AC
GDP GTP
GTP
GDP
cytosol
ATP cAMP + PPi
 &  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.
hormone
signal
outside
GPCR
The complex
of  & 
subunits G,
inhibits G.
plasma
membrane

AC
GDP GTP
GTP
GDP
cytosol
ATP cAMP + PPi
The sequence of events by which a hormone
activates cAMP signaling:
1. Initially G has bound GDP, and ,, & 
subunits are complexed together.
hormone
signal
outside
GPCR
plasma
membrane

AC
GDP GTP
GTP
GDP
cytosol
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).
hormone
signal
outside
GPCR
plasma
membrane

AC
GDP GTP
GTP
GDP
cytosol
ATP cAMP + PPi
3. Substitution of GTP for GDP causes another
conformational change in G.
G-GTP dissociates from the inhibitory  complex &
can now bind to and activate Adenylate Cyclase.
hormone
signal
outside
GPCR
plasma
membrane

AC
GDP GTP
GTP
GDP
cytosol
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  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 -arrestin, that promotes removal of
the receptor from the membrane by clathrinmediated endocytosis.
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 cascades:
 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 G, that is released
when G binds GTP is itself an effector that binds to
and activates other proteins.
 Cholera toxin catalyzes covalent modification of Gs.
• ADP-ribose is transferred from NAD+ to an arginine
residue at the GTPase active site of Gs.
• ADP-ribosylation prevents GTP hydrolysis by Gs .
• The stimulatory G-protein is permanently activated.
 Pertussis toxin (whooping cough disease) catalyzes
ADP-ribosylation at a cysteine residue of the
inhibitory Gi, making it incapable of exchanging GDP
for GTP.
• The inhibitory pathway is blocked.
 ADP-ribosylation is a general mechanism by which
activity of many proteins is regulated, in eukaryotes
(including mammals) as well as in prokaryotes.
ADP
ribosylation
H
O
C
protein
NH2
O
+
N

O P O CH2 O
H
H
H
H
OH
OH
NH2
O
N
(CH2)3
NH
C
O
NH

O P O CH2 O
H
H
H
H
OH
OH
NH2
O
N

N
O P O CH2 N
O
O
H
H
H
H
+
NAD
OH
OH
(nicotinamide
adenine
dinucleotide)

O P O CH2
O
(CH2)3
H
NH
NH2
N
N
NH2+
+
N
H
N
O
O
C
N
H
H
OH
H
OH
H
protein
Arg
C
residue
NH2+
ADP-ribosylated
protein
NH2
nicotinamide
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 sheet.
Three switch domains
GTPS
have been identified, that
change position when GTP Inhibitory G
substitutes for GDP on G.
These domains include residues adjacent to
the terminal phosphate of GTP and/or the Mg++
associated with the two terminal phosphates.
O
GTP hydrolysis
N
NH
H
H
O
O

O
P
O
O
O
P
O
N
O
O
P
O
CH2
O
H
N
NH2
O
H
H
OH
H
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
G - side view of -propeller
G – face view of -propeller
The  subunit of the heterotrimeric G Protein has a
-propeller structure, formed from multiple repeats
of a sequence called the WD-repeat.
The -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 neurons.
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.
Most GTP-binding
proteins depend on
helper proteins:
GAPs, GTPase
Activating Proteins,
promote GTP
hydrolysis.
protein-GTP (active)
GDP
GEF
GTP
GAP
Pi
protein-GDP (inactive)
A GAP may provide an essential active site residue,
while promoting the correct positioning of the
glutamine residue of the switch II domain.
Frequently a (+) charged arginine residue of a GAP
inserts into the active site and helps to stabilize the
transition state by interacting with () charged O
atoms of the terminal phosphate of GTP during
hydrolysis.
protein-GTP (active)
GDP
GEF
GTP
GAP
Pi
protein-GDP (inactive)
 G of a heterotrimeric G protein has innate capability
for GTP hydrolysis.
It has the essential arginine residue normally
provided by a GAP for small GTP-binding proteins.
 However, RGS proteins, which are negative
regulators of G protein signaling, stimulate GTP
hydrolysis by G.
protein-GTP (active)
GDP
GEF
GAP
GEFs, Guanine
GTP
Pi
Nucleotide Exchange
protein-GDP (inactive)
Factors, promote
GDP/GTP exchange.
 An activated receptor (GPCR) normally serves
as GEF for a heterotrimeric G-protein.
 Alternatively, AGS (Activator of G-protein
Signaling) proteins may activate some
heterotrimeric G-proteins, independent of a
receptor.
Some AGS proteins have GEF activity.
Phosphatidylinositol Signal
Cascades
O
O
R1
C
H2 C
O
O
C
CH
H2 C
R2
O
O
P
O
O
OH
2
phosphatidylinositol
H
H
1
6
H
OH
OH
H
OH
5
H
3
H
4
OH
Some hormones activate a signal cascade based
on the membrane lipid phosphatidylinositol.
O
O
R1
C
H2C
O
O
C
CH
H2C
R2
O
O
P
O
O
OH
2
H
PIP2
phosphatidylinositol4,5-bisphosphate
H
1
H
OH
3
H
6
OH
H
4
OPO32
5
H
OPO32
Kinases sequentially catalyze transfer of Pi from
ATP to OH groups at positions 5 & 4 of the inositol
ring, to yield phosphatidylinositol-4,5bisphosphate (PIP2).
PIP2 is cleaved by the enzyme Phospholipase C.
Different isoforms of
Phospholipase C
have different
regulatory domains,
& thus respond to
different signals.
A G-protein, Gq
activates one form
of Phospholipase C.
O
O
R1
C
H2C
O
O
C
CH
H2C
cleavage by
Phospholipase C
R2
O
O
P
O
O
OH
2
H
PIP2
phosphatidylinositol4,5-bisphosphate
H
1
6
H
OH
OH
H
3
H
OPO32
5
H
4
OPO32
When a particular GPCR (receptor) is activated, GTP
exchanges for GDP. Gq-GTP activates
Phospholipase C.
Ca++, which is required for activity of Phospholipase C,
interacts with () charged residues & with Pi moieties of
the phosphorylated inositol at the active site.
OPO32 H
OH
2
H
1
6
H
OH
OH
H
3
H
OPO32
O
5
H
4
OPO32
IP3
inositol-1,4,5-trisphosphate
O
R1
C
H2C
O
O
C
R2
CH
H2C
OH
diacylglycerol
Cleavage of PIP2, catalyzed by Phospholipase C,
yields 2 second messengers:
 inositol-1,4,5-trisphosphate (IP3)
 diacylglycerol (DG).
Diacylglycerol, with Ca++, activates Protein Kinase
C, which catalyzes phosphorylation of several
cellular proteins, altering their activity.
Ca++
Ca++-release channel
IP3
Ca
ATP
calmodulin
Ca
++
endoplasmic
reticulum
Ca++-ATPase
++ ADP + Pi
IP3 activates Ca++-release channels in ER
membranes.
Ca++ stored in the ER is released to the cytosol,
where it may bind calmodulin, or help activate Protein
Kinase C.
Signal turn-off includes removal of Ca++ from the
cytosol via Ca++-ATPase pumps, & degradation of IP3.
OPO32 H
OH
OPO32
OH
OH
H
H
OH
H
H
OPO32
H
IP3
(3 steps)
H
OH
OH
H
OH
OH
H
+ 3 Pi
H
H
H
OH
inositol
Sequential dephosphorylation of IP3 by enzymecatalyzed hydrolysis yields inositol, a substrate for
synthesis of PI.
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.
O
O
R1
C
H2C
O
O
C
CH
H2C
R2
O
O
P
O
O
phosphatidylinositol3-phosphate
OH
2
H
H
1
6
OH
H
OPO32 H
3
H
4
OH
5
H
OH
The kinases that convert PI (phosphatidylinositol)
to PIP2 (PI-4,5-P2) transfer Pi from ATP to OH at
positions 4 & 5 of the inositol ring.
PI 3-Kinases instead catalyze phosphorylation of
phosphatidylinositol at the 3 position of the inositol
ring.
O
O
PI-3-P, PI-3,4-P2,
PI-3,4,5-P3, and
PI-4,5-P2 have
signaling roles.
R1
C
H2C
O
O
C
CH
H2C
R2
O
O
P
O
O
phosphatidylinositol3-phosphate
OH
2
H
H
1
6
OH
H
OPO32 H
3
H
4
OH
Head-groups of these transiently formed lipids are
ligands for particular pleckstrin homology (PH) &
FYVE protein domains that bind proteins to
membrane surfaces.
Other protein domains called MARKS are (+)
charged, and their binding to () charged headgroups of lipids like PIP2 is antagonized by Ca++.
OH
5
H
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.
Signal complexes are often associated with lipid
raft domains of the plasma membrane.
Scaffold proteins as well as signal proteins may
be recruited from the cytosol to such membrane
domains in part by
 insertion of lipid anchors
 interaction of pleckstrin homology or other
lipid-binding domains with head-groups of
transiently formed phosphatidylinositol
derivatives, such as PIP2 or PI-3-P.
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.