Chapter 14 Signal-transduction pathways

[proteins in cell membrane]

Delivery

[2 messenger]

Hormones, Aromas (volatiles) sense1 messenger[threshold conc.][ligand]

changes in enzyme activity, gene expression, or ion-channel activity

metabolic pathways

Branch / feedback / terminate

Common second messengers

(1) Free to diffuse to other compartments of the cell

(2) Significantly amplified signals

(3) Common 2º messengers utilized

— cross talk: opportunities and potential problems

[NO]

H2O2

cADP ribose

Hormones:

Signal transduction

Signal molecules

nonpolar large and polar

through membrane bind to receptor

bind to protein alter receptor structure

interact with DNA [2° messengers]

modulate gene expression protein P / de-P

biochemical reaction

intracellular

steroidsligand,

primary messenger

Type 1: Seven-transmembrane-helix (7TM) receptor

–– serpentine receptor

ca. 50 % therapeutic drugs

rhodopsin

C-terminal and cytoplsmic loop conformation changes

Guanyl nucleotide-binding protein (G protein)

7TM

G-protein-coupled receptors

(GPCRs)

ATP cAMP

an intermediary in signal transduction from 7TM

a heterotrimeric G proteins

G protein: a heterotrimer

: bind the nucleotide (activated and unactivated state): a seven-bladed propeller: a pair of -helices

p. 381

G-protein-coupled receptors (GPCRs)(02)

Activated G proteins transmit signals by binding adenylate cyclaseATP cAMP

The epinephrine receptor signal-transduction

pathway [cAMP] stimulate ATP production for muscle contrac

tion

enhance the degradation of storage fuels

increase the secretion of acid by gastric mucosa

lead to the dispersion of melanin pigment granules

diminish the aggregation of blood platelets

induce the opening chloride channel

mediated by protein kinase A(PKA)

target protein—ser/thr-P

cAMP- response element binding (CREB) protein, in nucleus

- a transcriptional activator

Serotonin

Close K+ channel

cAMP- response element binding protein

1. Resetting G subunit: a intrinsic GTPase activity, spontaneo

us

seconds ~minutes

A build-in clock

Mechanism?

How to terminate the signal transduction?

Signal termination

cytosol

dep. hormone conc.

Specific

(for C-terminal

and occupied)

diminishes its ability to activate G proteins

Phosphatidyl inositol bisphosphate hydrolysis two messengers

Vasopressin

7TM

Gq

Phospholipase C

Cleavage PIP2

IP3 (soluble form)

DAG (insoluble form)

A1

A2

C

D

1,4,5

DAG and IP3

work in tandem

IP3 open channels to release Ca2+

3IP3 + IP3 -gate channel

Ca2+ release (from ER, SR)

Smooth muscle contraction

Glycogen breakdown

Vesicle release

Early fertilization

a short-lived messenger

(nM)

(02)

Diacylglycerol metabolism

Arachidonate

Prostaglandin H2

phosphorylated

hydrolyzed

(02)

Ca2+ — a ubiquitous cytosolic messenger

The reasons for Ca2+ mediate many signaling processes1. Fleeting changes in [Ca2+] are readily detected

The low level of [Ca2+]cyto can be easily and abruptly

raised for signaling purposes 100 nM

avoid insoluble compound formation

(an apparent drawback is in fact an advantage)

is via Ca2+-ATPase and Na+- Ca2+ exchanger (02)

2. Ca2+ can bind tightly to proteins and result in

significant conformational changes coordinated with six to eight oxygen atoms

from proteins or water

6 O from aa/protein

1 O from water

Plagiarism

How to detect or monitor the variation of [Ca2+] in real time

- The fluorescent calcium-binding dye

Red: high

Blue: low

Calcium-specific reagents (02)

ionophores

raise the cytosolic Ca2+ level reduce the unbound Ca2+ level

EDTA: ethylenediamine tetraacetic acid

Calmodium – a calcium sensor

¤ a 17-kd with 4 calcium-binding sites

¤ is activated when [Ca2+]cyto 500 nm

¤ a member of EF-hand protein family, a calcium binding motif

- a helix-loop-helix unit

¤ Parvalbumin: vitamin D3-dependent Ca2+ binding proteins

thumb

forefinger

Calmoduline-dependent protein kinase (CaM kinase)– recognize positively charged,

amphipatic helix

Calcium bind

calmodium conformational changes

A pair of EF-hand motifs

a flexible helix

expose hydrophobic surfaces that can be used to bind other proteins

calcium + calmodulin

CaM kinase Ca2+-ATPase pump

active target protein [Ca2+]cyto level decrease

signal propagation signal termination (the memory of a

previous calcium pulse)

(02)

Type 2: Insulin signaling

receptors that include protein kinase as part of their structures

receptor is a dimer of two identical units

each unit: - and -chain linked by a disulfide bond

-chain-chain

2 inter- and 1-intra-chain disulfide bond

One insulin binding on the outside of the cell

A membrane-associated kinase within the cell is activated;

cross-phosphorylation

Protein kinase A: Ser/Thr P

in subunit

3 tyr residues in activation loop of subunit

IRS : insulin-receptor substrate

a series of membrane-anchored molecules

IRS : insulin-receptor substrate

IRS1/IRS2, act as adaptor proteins

N-terminal: Pleckstrin homology domain, binds phosphoinositide lipids

phosphotyrosine-binding domain:

Tyr-X-X-M sequence: are phosphorylated by the receptor tyrosine kinase

Met

IRS phosphoinositide 3-kinase:

a lipid kinase, 110 kd catalytic subunit and 85 kd regulatory subunit

containing a SH2 domain: Src homology 2, recognize the

phosphotyrosine residues in the IRS, via two Arg residues that are

conserved in all SH2 domain

phosphoinositide 3-kinase PIP3

PIP3-dependent protein kinase

Akt: a kind of protein kinase, is not membrane anchored

Glucose transporters (GLUT4)

Stimulate glycogen synthesis

Membrane-anchor molecules

Amplicification/ termination

phosphatase phosphatase

phosphatase

Type 3: EGF (epidermal growth factor) signaling

stimulate the growth of epidermal and epithelial cells

a receptor tyrosine kinase, a 6 kd polypeptide

3 intrachain disulfide bonds

EGF receptor structure

EGF receptor:

is a dimer of two identical units, but exist as monomers until EGF

ligands bind to them

each monomer binds a EGF molecule in its extracellular domain

each EGF molecule lies far away from the dimer interface

a dimerization arm from each monomer that reaches out and inserts

into a binding pocket on the other monomer

If EGF is absent?

binds to a part of within the same monomer

Once EGF present,

Change into a active conformation

A constitutive active form ?

Her 2 receptor, 50 % identical in aa sequence

with the EGF receptor and has the same domain

structure

Her 2 is overexpressed in some cancers

EGF phosphorylation:

also like insulin receptor, cross-phosphorylation of one unit by another unit

within a dimer, but

its carboxyl - terminal tail containing tyrosine rich (5 residues)

the kinase itself is an active conformation without phosphorylation

Dimerization C-terminal region on one receptor into the active site

of its partner’s kinase

Grb-2: an adaptor protein

SH2 domain phosphotyrosine residues of receptor

SH3 domain proline-rich region of Sos

Sos: a guanine-nucleotide-exchange factor (GEF)

Ras: small G proteins, small GTPase

localized to the inner surface of plasmamembrane

two

phosphatase

GTPase-activating proteins (GAPs)

G proteins vs. small G proteins(divergent evolution)

G proteins small G proteins

30-35 kd 20-25 kd

heterotrimer monomer (similar to G)

7TM dimerization

GTPase act. GTPase act. (low)

GTPase-activating proteins (GAPs):

facilitate GTP hydrolysis Sos + GAPs adjust small G cycle

ras mutation cancer

14.4 Many elements recur with variation

in different signal transduction pathways

Protein kinases are central

Second messengers

Specialized domains

pleckstrin homology domains: interact with lipids PIP3

SH2 domains: interact with the phosphorylated tyrosine residues

Some virus induced cancer– to understand the signal-transduction proteins and

pathwaysRous sarcoma virus: a retrovirus, a oncogenic RNA virusviral sarcoma (v-src): oncogene [A cancer-causing gene; any of several m

utant genes that cause cells to exhibit rapid, uncontrol proliferation.] cellular sarcoma (c-src): proto-oncogene, does not induce cell transformat

ion

a. SH2 bind to tyr-P of C-terminalb. The linker between SH2 and protein kinase is bounded by SH3 c-Src inactive

v-Src: 11 aa of C-terminal, lack Y residue

always active

Biology/chemical/physical factors

c-Src

19 aa

Ras: a small G protein or GTPase– localized to the inner surface of plasmamembrane

The small G proteins

Three 21-kd Ras proteins in mammalian cells

H-Ras: Harvey rat sarcoma

K-Ras: Kirsten rat sarcoma A loss of the ability to hydrolyze GTP

N-Ras: Neuroblastoma rat sarcoma continue on

Tumor-suppressor genes (contribute to cancer development):

to develop cancer only when both copies of the genes normally present in a cell

are deleted or otherwise damaged.

e.g., genes for some of the phosphatase

Monoclonal antibodies utilization:

inhibit the signal transduction in activated tumor formation

In some human epithelial cancers, such as breast, ovarian, and

colorectal cancers, overexpressed the epidermal-growth-factor receptor (EGFR)

Monoclonal Ab offend receptor

e.g., Cetuximab, target a receptor tyrosine kinase

Trastuzumab (Herceptin): inhibit Her2 overexpressed in breast cancers

Protein kinase inhibitor– a potential anticancer drugs

Chronic myologenous leukemia (CML)

chromosome defect:

the translocation between chromosome 9 and 22

(reciprocally)

Bcr-Abl fused protein:

overexpress kinase activity and is not regulated appropriately

STI-571: a specific Bcr-Abl kinase inhibitor

encode tyrosine kinase

To understanding the signal-transduction pathways is leading to conceptually new disease treatment.

Metabolism disease

Choleragen

a cholera toxin from Vibrio cholera (G -) two functional units: subunit B: bind to GM1 gangliosides of intestinal epithelium (p. 738) subunit A: enters the cell, catalyze the covalent modification of Gs

proteinGs + subunit A Gs-Arg-ADP-ribose

stabilize Gs-GTP form (perpetually stimulation)

activate adenylate cyclase

[cAMP] activate protein kinase A

open Cl- channel / inhibit Na+-H+ exchanger

NaCl and H2O loss

Treatment consists of rehydration with a glucose-electrolyte solution.

Pertussis toxin

is secreted by Bordetella pertussis

Gi + pertussis Gi-ADP-ribose

reduced Gi-GTP affinity

inhibit adenylate cyclase

[cAMP] close Ca2+ channels and open K+ channels

Altered G-protein activity caused diseases

(02)

96T

(191)

96T (192)

STAT5(signal transducers and activators of

transcription)–– a regulator of gene expression

–– is phosphorylated by JAK2

recirpocal interaction

(02)

97T

Which of the event is important for epidermal growth factor signaling?(A) ADP-ribosylation (B) Farnesylation (C) Mono-oxygenation (D) Peroxidation (E) Glycosylation (94, 台大 ) p. 284

Nelson p. 474

96C