Rao for CMB Friday

See his slides.
Rao Notes 1/5/06

Signal Transduction

Assignment will be given Monday to be due the following Monday.
1
Hormones and neurotransmitters are considered extracellular signals.
Obj 4: We will need to know details of well-characterized pathways.
He will point out what is important and what is not.
2
You have a signaling cell and a responding cell. A cascade of reactions leads to cellular response.
Types of signals:
Signal can be a secreted molecule or a change in concentration of a metabolite or nutrient. Can also be a physical signal like light, touch, heat, etc.
Operational distance may vary from system to system. Endocrine- produced in signalling cell or organ to act on distant organ.
Paracrine- act on neighboring cell.
Autocrine- signalling product secreted, acts on same cell. Ex: tumor cells secrete growth factors to act on same cell to keep cells proliferating.

Receptor types are specific to the signal.
Receptors are classified based on ligand or mechanism of signal transduction.
Some receptors are membrane bound protein interacting with a membrane-bound receptor (Notch).
Some penetrate into cell for intracellular receptors (steroids)
Once receptor activated, pathway may be short (activate proteins on surface, like ion channels- GPCR quick response through ion channel). Or it may involve 2nd messenger like cAMP, etc listed on p2.
Gap junction can transfer signal one cell to another without activating receptors on those cells.
2nd messengers activate through change in activity of preexisting proteins (post-translational modification) or by cascade of reactions leading to activation of gene in nucleus and production of new protein.
Cell responses are listed.
Process of conveying signal into cell response is called signal transduction.
3
slide not given.
Strategy in Study of Signal Transduction
1. What molecules are involved?What pathways?
2. When are signals activated? What is their timing? Are they transient or long-lasting or sustained?(kinetics)
3. Where is signal distributed or localized? Where is activated within the cell?
Common methods to answer these questions:
1. What
A. Use inhibitors.Design inhibitor specific to molecule- not always possible. May be selective (active at a specific concentration), but not specific. You have to screen the inhibitors that claim to be specific.
B. Interfering molecules are used for situations when activation involves translocation (Ex PKC to plasma membrane from cytosol- protein RACK is very specific. Add peptide, it goes between RACK and PKC to inhibit interaction. Signal is blocked by blocking translocation.
C. Downregulation
1. reduces expression of protein with antisense oligonucleotide (15-20 nucleotides specific for gene, design nucleotide)
2. siRNA
3. shRNA- transfect cells with construct to produce interfering RNA or use inducible vector. Sometimes cells may not grow if you turn off expression altogether.
D. Expression of Mutants
1. Dominant negative- can bind, but kinase activity is not there. IN PKCEta- eliminate lysine, cannot bind ATP, no activity. Competes with endogenous PKC.
2. C-Src is constitutively active by mutating inhibitory tyrosine so it cannot be phosphorylated to turn the molecule off.
2. When
A. Time course of Activation- see high levels by antibodies that recognize phosphorylated or active forms. Can see time course of activation.
B. Transient vs persistent: can determine cellular response
3. Where
A.Can prepare subcellular fractions or nuclear or mitochondrial fractions, immunoblot, see where molecule is
B.Use immunohistochemistry
C. Visualize in live cell with tag of fluorescence protein.
5
Know the above strategies.
Can use inhibition to block steps of cascade, interfere, reduce expression, , or express mutant forms and see how cellular response is altered. Can also tag with GFP to localize.
6
Shared principles
1. shown on slide
2. receptors are not uniformly distributed. With small number of receptors, response is made more efficient by clustering with adaptor proteins with common binding sites. Diagram shows anchoring to actin cytoskeleton. Caveolin can capture and cluster certain receptors in lipid rafts.
3. Many signalling paths use GTPase switch proteins. Guanine nucleotide exchange factor facilitates GDP-GTP exchange. 2 kinds: trimeric G proteins bind receptor directly. Monomeric G-proteins are indirectly activated. Other conserved proteins are kinases or phosphatases Y/S/T are amino acids with an OH group, so they can be phosphorylated. PKC and PKA are S/T kinases. PTK is Y kinase.

7
cAMP signaling Pathway- know details.
cAMP is produced by adenylyl cyclase which converts ATP to cAMP. Cyclase is regulated by receptors which activate or inactivate. Learn the diagram.
PKA has catalytic and regulatory subunits. Normally C is bound to pseudosubstrate on R subunit. cAMP binds to sites on regulatory subunit and conformational change releases C subunit. Enzyme phosphorylates target protein.
Several signals induce cAMP or activate PKA. AKAP binds PKA and translocates it to specific target sites for specific responses. PKA isoforms can also be expressed in different cells for different responses.
Clinically relevant features: choleratoxin, etc listed on slide. Know role of PKA and how it is regulated.
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Phosphoinositide related signaling pathway
Dynamic regulatory function.
PIP2 has high turnover. Phospholipase C breaks phosphoester bond to form DAG and IP3. Both are second messengers.
PLC can be regulated by GPCR or RTKs. Turn on/off or activate/deactivate by phosphorylation. Ca binds with PKC or has other signalling mechanisms. Not all isoforms of PKC require Ca. DAG can also attract isoforms of PKC.

Phosphorylation on position 3- bind to pleckstrin homology(PH) domains for protein-protein interaction. Can activate protein kinase B.
Actin binding proteins have PH domain and are usually near membrane. PLC releases them. Phospholipids can bind proteins in an inactive form which are released later.