Cell Communication Single cell Multicellular organism

Budding yeast cells responding to mating factor One haploid cell Another haploid cell Sexual mating (requires many downstream signal pathways to be activated)

GENERAL PRINCIPLES OF CELL COMMUNICATION Extracellular signal molecules bind to specific receptors

Extracellular signal molecules can act over either short or long distance

Autocrine signaling can coordinate decision by groups of identical cells “Community effect” in early development In tumor biology---cancer cells stimulate their own proliferation

Gap junctions allow signaling information to be shared by neighboring cells Ca 2+, cAMP etc. but not for proteins or nucleic acids Intracellular electrodes, small water-soluble dyes Connexin 43 deficiency --- abnormal heart development

Each cell is programmed to respond to specific combinations of extracellular signal molecules

Different cells can respond differently to the same extracellular signal molecules

The concentration of a molecule can be adjusted quickly only if the lifetime of the molecule is short

Nitric oxide gas signals by binding directly to an enzyme inside the target cell Nitroglycerine --- angina Viagra --- PDE inhibitor CO

Nuclear receptors are ligand-activated gene regulatory proteins

Ligand-binding domain

The three largest classes of cell-surface receptor proteins are ion-channel-linked, G-proteins-linked, and enzyme-linked receptors

Most activated cell-surface receptors relay signals via small molecules and a network of intracellular signaling proteins

Some intracellular signaling proteins act as molecular switches 2% of human genes Monomeric GTPase Trimeric GTPase

Signal integration by protein phosphorylation

Intracellular signaling complexes enhance the speed, efficiency, and specificity of the response

Complex forms transiently

Interactions between intracellular signaling proteins are mediated by modular binding domains

PDZ Domain Domain binding and function: PDZ domains bind to the C-terminal 4 – 5 residues of their target proteins, frequently transmembrane receptors or ion channels. These interactions can be of high affinity (nM K d ). The consensus binding sequence contains a hydrophobic residue, commonly Val or Ile, at the very C-terminus. Residues at the – 2 and – 3 positions are important in determining specificity. PDZ domains can also heterodimerize with PDZ domains of different proteins, potentially regulating intracellular signaling. In addition to engaging in protein- protein interactions, several PDZ domains including those of syntenin, CASK, Tiam1 and FAP are capable of binding to the phosphoinositide PIP 2. PIP 2 -PDZ domain binding is thought to control the association of PDZ domain-containing proteins with the plasma membrane. Structure Reference: Doyle, D.A. et al. (1996) Cell 85(7), 1067 – The third PDZ domain from PSD-95.

Binding Examples: PDZ domain proteins Binding partners domain binding sites Post-synaptic Density Protein 95 (PSD-95) NMDA receptor B via PDZ1 and PDZ2 of PSD-95 – IESDV-COOH Post-synaptic Density Protein 95 (PSD-95) Kvl1.4 Shaker-type K + channel via PDZ1 and PDZ2 of PSD-95 – VETDV-COOH Post-synaptic Density Protein 95 (PSD-95) Neural Nitric Oxide Synthase (nNOS) via PDZ2 PDZ/PDZ interaction

Enriched in cholesterol and glycolipids Lipid raft c-Src tyrosine kinase

Cells can respond abruptly to a gradually increasing concentration of an extracellular signal Chicken oviduct cells Stimulated by estradiol effector/target : 1~16 maximal activation

One type of signaling mechanism expected to show a steep thresholdlike response

A cell can remember the effect of some signals Signals trigger muscle cell determination Autophosphorylation of Ca 2+ /CaM-kinase II

Cells can adjust their sensitivity to a signal

SIGNALING THROUGH G-PROTEIN-LINKED CELL-SURFACE RECEPTORS 1. The largest family of cell-surface receptors 2. 5% of the C. elegans genes 3. Signal molecules: hormones, neurotransmitters and local medicators 4. Rhodopsin-light receptor 5. Genome sequencing --- vast numbers of new family members 6. Major targets for drug discovery

Trimeric G proteins disassemble to relay signals from G-protein-linked receptors Transducin-G protein in visual transduction

The disassembly of a activated G-protein into two signaling components

The switching off of the G-protein  subunit by the hydrolysis of its bound GTP RGS proteins --- regulators of G protein signaling, act as  subunit-specific GTPase activating proteins (GAPs) ~25 RGS proteins in the human genome

Some G-proteins signal by regulating the production of cyclic AMP Nerve cell culture, preloaded with a fluorescent protein that changes its fluorescence when it binds to cAMP. >10 -6 M~5 X M (Science 260: , 1993)

cAMP-dependent protein kinase (PKA) mediate most of the effects of cyclic AMP Role of cAMP, PKA in glycogen metabolism

How gene transcription is activated by a rise in cAMP concentration (CRE, cAMP response element) Role of protein phosphatases?

Some G-proteins activate the inositol phospholipid signaling pathway by activating phospholipase C- (<1% of total phospholipids)

The two branches of the inositol phospholipid pathway

Ca 2+ functions as a ubiquitous intracellular messenger Ca 2+ signaling in fertilization of starfish, detected by Ca 2+ -sensitive fluorescence dye

The main ways eucaryotic cells maintain a very low concentration of free Ca 2+ in their cytosol

The frequency of Ca 2+ oscillations influences a cell’s response In a liver cell

Ca 2+ /calmodulin-dependent protein kinases (CaM-kinases) mediate many of the actions of Ca 2+ in animal cells The structure of Ca 2+ /calmodulin A peptide derived from CaM-Kinase II

The activation of CaM-kinases II ~2% of total mass in some brain regions, especially in synapses It can function as a molecular memory device --- (1)Learning defect (where things are in space) in mutant mice that lack the brain-specific subunit of CaM-kinase II (2) Same defect also observed in mutant mice that have their CaM-kinase II mutated at the autophosphorylation site

CaM-kinases II as a frequency decoder of Ca 2+ oscillations CaM-kinase II is immobilized on a solid surface +a brain protein phosphatase +repetitive pulse of Ca 2+ /calmodulin at different frequency Kinase activity assay What a nice experiment it is!

Smell and vision depend on G-protein-linked receptors that regulate cyclic-nucleotide-gated ion channels

Cyclic GMP A rod photoreceptor cell

The response of a rod photoreceptor cell to light

Extracellular signals are greatly amplified by the use of small intracellular mediators and enzymatic cascades Amplification in the light-induced catalytic cascade in vertebrate rods

G-protein-linked receptors desensitization depends on receptor phosphorylation

SIGNALING THROUGH ENZYME-LINKED CELL-SURFACE RECEPTORS Six classes: 1. Receptor tyrosine kinases 2. Tyrosine kinase-associated receptors 3. Receptorlike tyrosine phosphatases 4. Receptor serine/threonine kinases 5. Receptor guanylyl cyclases 6. Histidine-kinase-associated receptors

Activated tyrosine kinases phosphorylate themselves angiogenesis cell/axon migration

Three ways in which signaling proteins can cross-link receptor chains Monomeric vs. dimeric ligand

Inhibition of signaling through normal receptor tyrosine kinases by an excess of mutant receptors As a tool for determining normal function of receptor

Phosphorylated tyrosine serves as docking sites for proteins with SH2 domains

The binding of SH2-containing intracellular signaling proteins to an activated PDGF receptor determine the binding specificity

Ras is activated by a guanine nucleotide exchange factor GEF: guanine nucleotide exchange factor GAP: GTPase-activating protein In cells [GTP] > [GDP] ~10 fold

The activation of Ras by an activated receptor tyrosine kinase

The MAP-kinase serine/threonine phosphorylation pathway activated by Ras

The organization of MAP-kinase pathway by scaffold proteins in budding yeast

PI 3-kinase produces inositol phospholipid docking sites in the plasma membrane Cell division vs. cell growth PI 3 kinase is one of the major cell growth signaling transduces

The recruitment of signaling proteins with PH domains to the plasma membrane during B cell activation SH2 domain Mutation of BTK leads to severely deficiency in Ab production

The PI 3-kinase/protein kinase B signaling pathway can stimulate cells to survive and grow

Brief summarization

Signal proteins of the TGF- superfamily act through receptor serine/threonine kinases and Smads

Kinase catalytic domain ~250 amino acids

SIGNALING PATHWAY THAT DEPEND ON REGULATED PROTEOLYSIS 1.Notch 2.Wnt 3.Hedgehog 4.NF-kB

The receptor protein Notch is activated by cleavage In Drosophila, mutation in Delta leads to produce a huge excess of neurons at the expense of epidermal cells

The processing and activation of Notch by proteolytic cleavage Inhibit neural differentiation

Wnt proteins bind to Frizzled receptors and inhibit the degradation of -catenin (c-Myc protein)(APC, adenomatous polyposis coli, a tumor suppressor )

Multiple stressful and proinflammatory stimuli act through an NF-B-dependent signaling pathway inflammation development cancer