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Effects of Second Messengers

Inositol trisphosphate

Opens Calcium Channels

Binding to IP3-gated Channel

Cooperative binding

Diacylglycerol

Activates Protein Kinase C

Ca2+ is requiredPhosphorylation of

many target proteins

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The domain structures of protein kinase C isoforms

Interaction with phospholipids

Binds Diacylglycerol

Pseudosubstrate Sequence

• Resemble the substrate sequence• Contains

A-R-K-G-A-L-R-Q-KSubstrate Sequence

X-R-X-X-(S,T)-Hyd-R-X

• Binds to the Enzyme’s Active Site.

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Termination of IP3 Signal

Lithium Ions,Used to treat some

psychological disordersInhibits IP3 recycling

IP3 is a Short-LivedMessenger

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Why Ca2+?A large difference in concentration

0.1μM

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+Ca2+

Ca2+

Ca2+

Ca2+Ca2+

Ca2+

Ca2+

Ca2+Ca2+

Ca2+

mM

Ca2+

Why Ca2+?

Ability to bind protein tightly

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Useful Tools in Studying the role of Calcium

• Ionophores– Introducing Calcium into the cell

• Calcium Chelators– Decreasing Calcium Concentration

• Fluorescent Chelators– Measuring Calcium Concentration

Calcium Binding Proteins

• Mediate the effects of Calcium• Many proteins

Calmodulin, Troponin C, Parvalbumin• Similar structures

– Rich in Asp and Glu– Several α helical segments– Binding site is formed by

Helix Loop Helix

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Helix E

Calmodulin

• Found in almost all eukaryotic cells• Consists of two globular regions

– Connected by flexible region– Each contains 2 EF hands– Four Ca2+ binding sites.

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Calmodulin changes conformation upon binding to Calcium

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4 Ca2+Calmodulin binds to Ca2+

which results in change in conformation

( Moving some hydrophobicresidues from

the inside to the outside of the domains)

Calcium-Calmodulin Complex can Bind to a large Number of Enzymes,Pumps and Target proteins including

Calmodulin-dependant Protein Kinase

Ca2+ ATP’ase Pump

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Ca2+ Transporter

• In sarcoplasmic reticulum– 80% of the membrane proteins– 10 membrane spanning helices– Ca2+ move against a large concentration gradient– 2 Ca2+ / ATP

Signal Transduction through Tyrosine Kinase

Hormone Binding

Dimerization of the receptor

Auto phosphorylation of the receptor

Phosphorylation of the target proteins

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Some Hormones that use Tyrosine Kinase

• Growth Hormone• Insulin• Epidermal Growth Factor • Platelet-derived growth Factor

Growth Hormone

• Monomeric Protein• 217 Amino Acids• Compact Four-helix Bundle

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Growth Hormone Receptor

• 638 A.Acid– Membrane Spanning Protein– Extracellular Domain ≈250 A.A– Single Membrane-Spanning Helix– Intracellular Domain 350 A.A

• Monomeric when not bound to hormone• Dimeric when bound to hormone

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GrowthHormone

Binding of one molecule of growth hormone

Dimerization of the receptor

Each Intracellular Domain is associated with a protein kinase called

Janus Kinase 2

Interaction with

membrane

Binds peptides that contain

Phosphotyrosine

protein kinaseprotein kinase-like

Janus

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Receptor dimerization brings two JAKs together

Each Phosphorylates key residues on the other

Activated JAK 2 can Phosphorylate other substrates

• STAT 5– Signal transducer and activators of transcription

• Regulator of transcription

• STAT5 Phosphorylation Dimerization

Binding to specific DNA sites

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STAT is phosphorylated on a tyrosine residue near the carboxyl terminus

Phosphorylated tyr binds to SH2 domain of another STAT 5 molecule

Activated JAK 2 can Phosphorylate other substrates (cont.)

• Phosphorylation of the Receptor

– Association with JAK 2

– Association with other proteins in the signal transduction pathway

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Signal-Transduction Cascades - 3

•Tyrosine kinase and receptor dimerization•Signaling Pathways and Cancer

Receptor dimerization brings two JAKs together

Each Phosphorylates key residues on the other

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Tyrosine Kinase is Part of some receptors

• Epidermal Growth Factor Receptor– Monomeric (inactive)– EGF binding Dimerization Cross

Phosphorylation Activation• Insulin Receptor

– Dimer of 2 αβ pairs– Insulin Binding ››› Activation of the Kinase

Do these receptors transfer information across the membrane in the same way?

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Is dimerization alone sufficient for activation of EGF receptor?

• Synthesis of a gene that encoded a chemeric receptor– Extracellular (insulin)– Intracellular (EGF)

• EGF receptor and insulin receptor use a common mechanism for transmitting information across membrane

Epidermal growth factor signaling pathway

• Grb-2 binds phosphorylated EGF receptor↓↓

Binding to a protein called Sos↓↓

Activation of Ras↓↓

Activation of specific Protein Kinases

GTPGDP

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Ras is a member of small G proteins family

• Monomeric• Exist in two forms

– GDP bound «-----» GTP bound• Smaller than G proteins• Have GTPase activity• Many similarities in structure and mechanism

with Gα

• Include several groups or subfamilies• Major role in growth, differentiation, cellular

transport, motility etc…

SH2

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Defects in Signaling Pathways Can Lead to Cancer and Other Diseases

• Cancer, is characterized by uncontrolled or inappropriate cell growth,

• Can be caused by certain viruses• Can be associated with defects in signal-

transduction proteins;failure of signal transduction process

Rous sarcoma virus (in Chicken) carries a gene called v-src

• Oncogene• Encode a tyrosine kinase protein• Similar protein is found in cells called c-Src

• Small differences in the amino acid sequences between the proteins

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Impaired GTPase activity can lead to cancer in human

• Mammalian cells contain 3 Ras proteinsMutation

Loss of ability to hydrolyze GTPRas is locked in “ON” position

continuous stimulation of growth

Cholera and Whooping Cough Are Due to Altered G-Protein Activity

• The cholera toxin: protein composed of two functional units– B subunit: binds to GM1 gangliosides of the intestinal

epithelium – A catalytic subunit: enters the cell. – A subunit catalyzes the covalent modification of a Gαs

protein: – Attachment of an ADP-ribose to an arginine residue.– Stabilization of the GTP-bound form of Gαs, – The active G protein, activates protein kinase A.– Openining of chloride channels

• Excessive loss of NaCl and the loss of large amounts of water into the intestine.