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Down-regulation of Phosphoinositide-activated Second Messenger System. Off-signals: 1. IP 3 rapidly dephosphorylated by phosphatases. 2. DAG rapidly hydrolyzed. 3. Ca 2+ rapidly pumped out. 4. Ser/Thr phosphatases dephosphorylate PKC and CaM kinase targets.

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Down-regulation of Phosphoinositide-activated

Second Messenger System


1. IP3 rapidly dephosphorylated by phosphatases.

2. DAG rapidly hydrolyzed.

3. Ca2+ rapidly pumped out.

4. Ser/Thr phosphatases dephosphorylate PKC and CaM kinase targets.


Fertilization of an Egg by a Sperm Triggering an

Increase in Cytosolic Ca2+

Prior to fertilization, the starfish egg was injected with a Ca2+-sensitive fluorescent

dye. A wave of cytosolic Ca2+ (red), released from the ER, is seen to sweep across

the egg from the site of sperm entry.


Wave of Ca2+ release and PKC activation spreading from the site of artificial

activation of a Xenopus egg.

The egg was injected with calcium red (a fluorescent dye sensitive to the [Ca2+])

and a fusion protein, consisting of green fluorescent protein and PKC, which

produces green fluorescence when PKC is activated.


Protease-Activated Receptors (PARs):

GPCR’s activated by a novel proteolytic

mechanism: Protease forms a Michaelis

complex with its receptor/substrate (E•S) to

proteolytically cleave a small peptide from

the NH2-terminus generating a “NEW”


This “new” NH2-terminus serves as a

tethered peptide ligand, binding intramolec-

ularly to the body of the “receptor” to effect

transmembrane signaling.

Proteases are typically serine proteases.

Platelet Thrombin receptor structure: PAR1

Mechanisms of Platelet Activation and Control, Plenum Press, 1993


Features of PAR Amino-terminal Exodomains

PARs’ hirudin-like


Hirudin C-tail DFEEIPEEYLQ





Cleavage site

Tethered ligand domains


Cleavage of the receptor/substrate

by the protease results in

“Irreversible” activation

Irreversible Activation by Cleavage

“Reversible” activation can be

accomplished with a small

peptide mimicking the new


Reversible Activation by Peptide Mimetic


One Receptor Can Couple to More than One G protein

Four different receptors (R1-4) are shown.

R3 couples to both Gi and Gq


Modulation of Glycogen Synthase Activity by Phosphorylation

This enzyme contains nine separate sites in five designated regions susceptible to phosphorylation by

several different cellular protein kinases. Consequently, the activity of this enzyme can be modulated in

response to a variety of second messengers produced in response to different extracellular signals.

Therefore regulation is a matter of finely tuned modulation of the enzyme activity over a wide range and

not merely on/off switching.


Structural/functional Consequences of Lipid Modifications

TIBS 16:338, 1991

  • Part of the GPCR C-terminus is tethered to the membrane by palmitoylation through a thioester linkage to a conserved Cys.
  • The G protein  subunit has myristate attached via an amide linkage to an N-terminal Gly to effect membrane association.
  • The G protein subunit is incorporated into the membrane via a geranylgeranyl moiety attached via a thioether linkage to
  • a C-terminal conserved Cys.
  • Some G protein-coupled effectors are transmembrane proteins (e.g. ion channels and adenylyl cyclase) as shown here,
  • others (e.g. phospholipase C) may be peripheral membrane proteins.

Production of the 2nd Messenger – cGMP

The membrane-bound form

of guanylate cyclase is acti-

vated directly by hormone

binding to its receptor, e.g.

atrial natriuretic peptides.

The cytosolic form is a different

protein and is activated by nitric

oxide (NO).

Protein kinase G (PKG) is activated by cGMP.


Functional Domains of the ANF Receptor

Figure 21.44 – Devlin, Textbook of Biochemistry


I. Cell Signaling by Nitric Oxide (NO)

Most cells can produce NO; however, it appears to function primarily in three

broad categories:

1. via the endothelial cell to cause vascular (smooth muscle cell) relaxation;

2. during neurotransmission to facilitate CNS function;

3. in cell-mediated immune responses to facilitate immunologic function.

NO is produced via the enzyme complex, NO synthase (NOS), which deaminates

Arg to form citrulline and NO


II. Cell Signalling by NO

Basal production of NO required to control vascular tone and normal blood pressure.

NO is chemically unstable and decomposes in seconds!!!

NO is chemically unstable and decomposes in seconds!!!

NO-mediated intercellular communication:

Agonists such as acetylcholine, thrombin, histamine and bradykinin activate their various

receptors on vascular endothelial cells, leading to activation of NO synthase.

NO diffuses from the EC to the

smooth muscle cell (SMC)

Within the SMC, NO stimulates

production of cGMP via activation

of a soluble guanylate cyclase.

cGMP activates cGMP-dependent

kinases (PKG) with the end result

being SMC relaxation.

Nitroglycerin and nitroprusside,

well known for treating angina

pectoris function by producing

NO during their metabolism.


Second Messengers Involved in Intracellular Signaling

© 2000 by W. H. Freeman and Company. All rights reserved.


Eukaryotic Signal Transduction Systems Involving

Membrane Receptors (1-5) and Second Messengers (1-4)


Second Messengers Involved in Intracellular Signaling

© 2000 by W. H. Freeman and Company. All rights reserved.


Signaling via Enzyme-linked Cell Surface Receptors

Receptor Protein Tyrosine Kinases (RPTKs)

Receptors are characterized by : 1) a single membrane-spanning domain; 2) a large glycosylated

extracellular domain defining the ligand binding site; and 3) an intrinsic enzymatic activity

contained within the cytoplasmic domain (tyrosine kinase activity)


Families of Ligands for Enzyme-linked Receptors

Molecular Basis of Medical Cell Biology, 1998

Ligands for RPTKs



Ligand-induced RPTK Dimerization and Subsequent Phosphorylation

© 2000 by W. H. Freeman

and Company. All rights


Some ligands are monomeric, e.g. EGF. Ligand binding induces a conformational

change in the receptors  receptor dimerization.

Other ligands are dimeric and bring two receptors together directly.

Irrespective, subsequent to dimerization, the kinase activity of each subunit cross-

phosphorylates Tyr residues near the active site in the other subunit. Autophos-

phorylation of Tyr residues within other parts of the cytoplasmic domain ensues.



Function of Phosphorylated Tyrosines

© 2000 by W. H. Freeman and Company. All rights reserved.

Phosphotyrosines of dimerized receptor serve as high affinity binding sites

for “docking” and/or “activation” of intracellular signalling proteins.



Selected Intracellular Substrates and Targets of RPTKs

Proteins that bind to Tyr-PO4 residues must contain

SH2 or SH3 domains [Src(soluble cytoplasmic tyrosine kinase)

Homology domains]

  • SH2 - 1– aa stretch; SH3- 60 aa stretch
  • Crystallographic studies demonstrated that these domains
  • have a pocket into which a Tyr-PO4 residue would fit
  • Adapter proteins link other cytoplasmic proteins to the
  • activated receptor

Molecular Basis of Medical Cell Biology, 1998


Binding of Various Signal-transducing Molecules

to Different Phosphorylated Tyrosines in the PDGF receptor


**denote Tyr residues

that are phosphorylated

***the outlined area in

each bound signal trans-

duction molecule repre-

sents the SH2 domain



Binding pocket

for a specific

amino acid side


Binding pocket for


The SH2 Domain: A Compact

“Plug-in” Molecule

The 3-D Structure of an SH2 Domain


Phospholipase C is Coupled to Gq

Phospholipase C Binds to Phosphorylated RPTKs


Mechanism of Activation of PLC-1 by EGF or PDGF

  • Binding of EGF (or PDGF) to its receptor elicits autophosphorylation subsequent to
  • receptor dimerization. (b) The PLC-1 forms a complex with the EGF (PDGF) phosphorylated
  • receptor and the tyrosine residues 771, 783 and 1254 are phosphorylated, which (c) activates
  • PLC-1.. (TIBS 16:297, 1991)

PLC-, -, - Share Regions of Significant Homology (X and Y)

(G protein activated)

(Tyrosine-kinase activated)

(TIBS 16:297, 1991)

(TIBS 16:297, 1991)

TIBS 16:297, 1991


How Do Growth Factor Receptors Lead to Cell Proliferation?

The Activation of Ras by an Activated RPTK

The Grb-2 adaptor protein binds to a specific phosphotyrosine on the receptor

and to the Ras guanine nucleotide exchange factor (GEF), which stimulates Ras

to exchange its bound GDP for GTP


What are Ras proteins and how are they activated?


Molecular Biology of the Cell, 2002

Ras proteins are monomeric GTPases.

GEFs activate Ras by stimulating it to give up its GDP for GTP. The cytosolic

[GTP] is 10 times > [GDP], so Ras readily binds GTP once GDP has been ejected.

Ras-GAPs inactivate Ras by stimulating its GTPase activity and thus its hydrolysis

of GTP to GDP. The Ras-GAPs maintain most of the Ras protein (95%) in an

unstimulated state.



© 2000 by W. H. Freeman and Company. All rights reserved.

© 2000 by W. H. Freeman and Company. All rights reserved.

© 2000 by W. H. Freeman and Company. All rights reserved.

Conformational Changes Accompanying Ras Activation

**Sos is a GEF specific for Ras


The MAP-kinase Ser/Thr phosphorylation pathway activated by Ras

MAP; mitogen

activated protein




Ras activates MAP-KKK (Raf), which in turn activates MAP-KK (Mek) by phosphoryla-

tion of select Ser/Thr residues. Activated Mek activates MAP-K (Erk) in an analogous

manner. Erk in turn phosphorylates a variety of downstream proteins resulting in

changes in gene expression and protein activity causing complex changes in cell behavior.


Growth Factor Activation of its RPTK Leading to Cell Proliferation

Leading to



Insulin-dependent stimulation of glucose uptake

occurs through a Ras-independent pathway!

IRS binds/stimulates PI-3 kinase, which effects an, as yet,

ill-defined cascade of events  the translocation of addi-

tional glucose transporters (GLUT-4) to the cell membrane.

New Engl J Med 341:248, 1999

GLUT-4 Translocation to the Plasma Membrane


Signal Transduction, Oncogenes and Cancer

Tumor cells can express mutationally altered forms or levels of proteins involved in signal

transduction – e.g. altered growth factors, growth factor receptors, G proteins, nuclear

receptors, protein kinases, etc.

Tumor cells may contain a normal signal transduction protein, but in excessive amounts.

The responsible gene is called an Oncogene. Corresponding normal gene is a Protooncogene.

How do protooncogenes become oncogenes?

1. Once a cellular “protooncogene” becomes part

of a retroviral genome, it can undergo mutation,

producing an oncogene, which can transform a

normal cell subsequent to retroviral infection.

2. Spontaneous or chemically-induced (carcinogens)

mutations in a “protooncogene.”

3. E.g., ras genes altered in codon 12,13 or 61 have

been detected in  30% of spontaneous and chemic-

ally induced tumors in animals and humans.


Most ras oncogene proteins

lack GTPase activity.

Several mutations that

generate ras oncogenes

are positioned close to the

bound guanine nucleotide.


EGFR and Cancer

  • Several mechanisms by which the EGFR system
  • becomes oncogenic:
  • autocrine ligand loops
  • amplification or overexpression of the EGFR
  • receptor
  • deletions or mutations that render the receptor
  • constitutively active independent of ligands

Activities of EGFR are mediated

by several signal transduction

pathways. The best characterized

is the Ras/Raf/Mek/Erk pathway,

which leads to gene transcription

resulting principally in cell growth

and proliferation.



Most common mutation produces EGFRvIII, which has a

truncated extracellular region with a distorted ligand-

binding area. Although this mutant cannot bind ligands,

it is trapped in a partially activated state, unable to undergo

downregulation. [(+) ligand dependence; (-) ligand independence]


EGFR as a Molecular Target for Cancer Therapy


MoAbs to EGFR


Anti-EGFRvIII vaccine

Bispecific Abs

Immunotoxin conjugates

Ligand-toxin conjugates


Small molecular inhibitors

of EGFR Tyr kinase


Antisense oligos



PTKRs and Bi-directional Signaling: Eph receptors and Ephrin ligands

(Dodelet VC and Pasquale EB. Oncogene 19:5614-5619, 2000)

Ephrin ligands are membrane-bound, with membrane

association occuring through a GPI linkage (A) or as

transmembrane proteins (B).

Upon cell-cell contact, Eph receptors and ephrins engage

in a class specific manner (A-A; B-B), which leads to

receptor clustering, receptor activation and receptor

phosphorylation (shown for EphB), which in turn provides

docking sites for SH2 domain-containing signaling proteins.

Upon receptor binding, ephrin-B ligands also become

Phosphorylated on Tyr, via an unidentified associated

tyrosine kinase. It is unknown if SH2 domain-containing

proteins also dock to phosphorylated ephrin-B ligands.

Ephrin/Eph interactions have been shown to be important

in clot retraction mediated by activated platelets.


Tyrosine Kinase-associated Receptors

Receptor Protein Tyrosine Kinases


Families of Ligands for Enzyme-linked Receptors

Molecular Basis of Medical Cell Biology, 1998

Ligands for Tyrosine-kinase

associated receptors


Ligands Activating Tyrosine Kinase-Associated Receptors

Primarily involved in signaling between cells of the immune and hematopoietic systems, and

signaling from cells near a site of inflammation


Tyrosine Kinase-Associated Receptors

Redundant Signaling:

Many members of the cytokine family can

stimulate the same response in a cell

Molecular basis of redundancy involves

sharing of receptor transducing subunits

Receptors composed of two polypeptide chains:

Recognition subunit specific for and binds


Transducing subunit initiates signaling



The Interleukin-3 (IL-3) Receptor

“Common”  subunit transduces

signal, which is shared by the

IL-5 and GM-CSF receptors

Subsequent to ligand binding, the receptor is phosphorylated on select Tyr residues by a special

family of cytoplasmic Tyr kinases: Janus kinases (JAKs) of which there are four members (JAK1,

JAK2, JAK3 and TYK2). These kinases appear to reside in close proximity to receptors on the inner

surface of the membrane. These kinases also phosphorylate signaling molecules containing the

required SH2/3 domains that dock on the receptor.




Subsequent to ligand binding, the receptor is phosphorylated on select Tyr residues

by a special family of cytoplasmic Tyr kinases: Janus kinases (JAKs) of which there

are four members (JAK1, JAK2, JAK3 and TYK2). These kinases appear to reside

in close proximity to receptors on the inner surface of the membrane.


The “Gleevec” Story


(Signal Transduction Inhibitor 571)

Causes overactive

white blood cell


Chromosome Swap in Chronic Myelogenous Leukemia


The Story of Gleevec (STI-571):

The “Magic Bullet” for the Cure of CML (Chronic Myelogenous Leukemia)

and GIST (Gastrointestinal Stromal Tumor)

STI-571 is specific for the unphosphorylated “inactive” form of the fusion protein Bcr-Abl, a cytoplasmic

Tyr kinase known to be involved in cell proliferation signaling pathways (left). It does not interact with

the Src kinase family (right).


Intracellular Receptors:

Nuclear Receptor Superfamilies of Ligand-activated Transcription Factors

Ligands can be steroid hormones derived from cholesterol

(e.g. cortisol, aldosterone, sex hormones, etc.), and related hormones

(e.g. thyroid hormones, vitamin D3 and retinoic acid, etc.), which

typically circulate in plasma bound to specific transport proteins.


Cellular Accumulation of Ligand

  • interact with specific intracellular receptors;
  • target cell entry is through passive diffusion; the rate of entry is directly
  • proportional to the intracellular steroid concentration (autocrine regulation);
  • inside the cell, the ligand may a) bind to its specific receptor or b) undergo
  • metabolism;
  • receptor criteria: finite binding capacity; high affinity; specific;
  • receptor characteristics: oligomeric, phosphorylated proteins (50,000-200,000
  • daltons) with three functional domains;




Intracellular Receptors

Molecular Biology of the Cell, 2002

C-terminus provides unique ligand binding site and sites for protein dimerization.

N-terminus defines region essential for transcriptional activation.

Middle domain (80 residues) contains the DNA binding site; exhibits as much as 50%

homology with other receptors; may associate with proteins that modulate their function.

Formation of a receptor/ligand complex can occur in the cytosol or the nucleus and

typically leads to dimerization.


Estrogen Receptor Binding to DNA

The dimeric receptor protein has two -helical regions that bind to both ends of a symmetrical DNA

sequence (AGGT-CAXXXTGACCT), within the major groove.


Formation of a receptor/ligand complex can occur in the cytosol or the nucleus and

typically leads to dimerization of the occupied receptor.

Dimerized receptor binds to the hormone regulatory element (HRE, 8-15 base pairs)

on the target DNA molecule  changes in gene transcription


Positive and Negative Transcriptional Effects of Steroid Receptors

Binding of a receptor dimer imme-

diately adjacent to a transcription

factor leads to synergistic activa-

tion of transcription

Binding of a receptor dimer to a

negative hormone response ele-

ment may displace a positive

transcription factor.

Protein-protein interaction between receptor

dimer and a positive transcription factor

such as AP 1 may block AP 1/DNA binding

and repress the transcriptional response.

Figure 22.15 – Devlin, Textbook of Biochemistry


In a given cell type, the extent and type of

receptor expressed will define the hormone


Devlin, Textbook of Biochemistry


Schematic representation of the receptor system and its transformation upon binding

of agonist (hormone H) or antagonist (antihormone AH).

With H binding, the inhibitor, hsp 90, is released (due to a

a conformational change within the receptor) and can interact with

its target DNA leading to a specific effect.

With AH binding, hsp 90 continue to bind to the receptor very

tightly, thus no interaction with DNA can occur.


Steroid Receptors as Drug Targets



effective breast cancer therapy

RU 486:


effective contraceptive


NF-B Proteins: Latent Gene Regulatory Proteins

Pivotal to Most Inflammatory Responses

  • Proinflammatory cytokines, such
  • as TNF-, bind to their specific
  • membrane receptors and initiate
  • a pathway that activates NF-B,
  • normally sequestered in an
  • inactive state through association
  • with IB proteins.
  • A TNF-/receptor interaction
  • initiates of pathway that marks
  • IB for degradation.
  • Degradation of IB exposes a
  • nuclear localization signal on NF-B,
  • which move into the nucleus.
  • Once activated, NF-B turns on
  • the transcription of > 60 genes
  • that participate in inflammatory
  • responses.

How glucocorticoids suppress immune and inflammatory reactions

mediated by cytokines

NF-B stimulates the

ultimate production of

inflammatory cytokines

Tumor necrosis factor (TNF)

binding to its receptor leads to

the ultimate degradation of IB

IB binds to

and inhibits the

nuclear translo-

cation of NF-B.

Glucocorticoid induction of

IB synthesis through GC

binding to its intracellular

receptor and stimulating trans-

cription of the gene.

A glucocorticoid interaction with its receptor results in increasing the transcription

of the protein IB, which binds and inhibits the activity of NF-B, a a transcriptional

activator that stimulates transcription of genes for inflammatory cytokines.