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.

Fertilization of an Egg by a Sperm Triggering an Increase in Cytosolic Ca 2+ Prior to fertilization, the starfish egg was injected with a Ca 2+ -sensitive fluorescent dye. A wave of cytosolic Ca 2+ (red), released from the ER, is seen to sweep across the egg from the site of sperm entry.

Wave of Ca 2+ 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 [Ca 2+ ]) 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 NH 2 -terminus generating a “NEW” NH 2 -terminus This “new” NH 2 -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 sequences

Hirudin C-tail D

FEEIP

EEYLQ PAR1 38 L-DPR //

SFLLR-N

PNDK

YEPFW

EDEEK PAR3 PAR4 35 43 L-PIK //

TFRGA-P

PN-S

FEEFP

FSALE LPAPR //

GYPGQVC

ANDSDTLELPDSSR PAR2 33 S-KGR //

SLIGK-V

DGTSHVTGKGVTV 

Tethered ligand domains

Cleavage site 61 57 68 55

Cleavage of the receptor/substrate by the protease results in “Irreversible” activation “Reversible” activation can be accomplished with a small peptide

mimicking

the new NH 2 -terminus Irreversible Activation by Cleavage Reversible Activation by Peptide Mimetic

One Receptor Can Couple to More than One G protein

Four different receptors (R

1-4

) are shown. R

3

couples to both G

i

and G

q

Consensus Sequences for Protein Kinases

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.

Sites of Lipid Modification of G Protein Subunits

TIBS 16:338, 1991

Production of the 2 nd 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-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 Ligands for RPTKs

Molecular Basis of Medical Cell Biology, 1998

BASIC PRINCIPLES OF RPTK SIGNALING

Ligand-induced RPTK Dimerization and Subsequent Phosphorylation

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

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.

BASIC PRINCIPLES OF RPTK SIGNALING

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.

BASIC PRINCIPLES OF RPTK SIGNALING

Selected Intracellular Substrates and Targets of RPTKs

Proteins that bind to Tyr-PO 4

SH2

or

SH3

domains [

S

residues must contain rc(soluble cytoplasmic tyrosine kinase)

H

omology domains] • SH2 - 1– aa stretch; SH3- 60 aa stretch • Crystallographic studies demonstrated that these domains have a pocket into which a Tyr-PO 4 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 chain Binding pocket for phosphotyrosine

The SH2 Domain: A Compact “Plug-in” Molecule The 3-D Structure of an SH2 Domain

Phospholipase C



Phospholipase C



is Coupled to G

q

Binds to Phosphorylated RPTKs

Mechanism of Activation of PLC  1 by EGF or PDGF (a) 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?

GEF

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.

**

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

Raf Mek Erk

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 proliferation

THE INSULIN RECEPTOR IS UNIQUE

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 Extracellular MoAbs to EGFR MoAbs to EGFRvIII Anti-EGFRvIII vaccine Bispecific Abs Immunotoxin conjugates Ligand-toxin conjugates Intracellular Small molecular inhibitors of EGFR Tyr kinase Nuclear Antisense oligos Ribozymes

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.

Receptor Protein Tyrosine Kinases Tyrosine Kinase-associated Receptors

Families of Ligands for Enzyme-linked Receptors

Ligands for Tyrosine-kinase associated receptors Molecular Basis of Medical Cell Biology, 1998

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 cytokine.

Transducing subunit initiates signaling response.

“Common”  subunit transduces signal, which is shared by the IL-5 and GM-CSF receptors The Interleukin-3 (IL-3) 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. These kinases also phosphorylate signaling molecules containing the required SH2/3 domains that dock on the receptor.

BASIC PRINCIPLES OF TYROSINE KINASE ASSOCIATED RECEPTOR SIGNALING I 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.

BASIC PRINCIPLES OF TYROSINE KINASE ASSOCIATED RECEPTOR SIGNALING II

Src Tyr Kinases are Required for MAP Kinase Activation of Gene Transcription

The “Gleevec” Story STI-571 (Signal Transduction Inhibitor 571)

Causes overactive white blood cell division 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 D 3 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;

SPECIFICITY ACHIEVED BY THE PRESENCE OF THE RECEPTOR IN THE TARGET CELL

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.

Receptor Activation Subsequent to Ligand Binding

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.

The Conserved DNA Binding Domain in Steroid Receptors

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 sensitivity.

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 Tamoxifen:

anti-estrogen; effective breast cancer therapy

RU 486:

anti-progesterone; 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 Tumor necrosis factor (TNF) binding to its receptor leads to the ultimate degradation of I



B



inflammatory cytokines 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.