What is cell signaling? The Basics:• Cell signaling refers to how information or a message moves inside

of the cytoplasm of a cell.• Signaling occurs in response to a ligand that binds to a specific

receptor causing changes inside the cell.• Cell signaling can lead to:

– Muscle contraction– Changes in transcription/translation– Changes in protein secretion and protein activity– Apoptosis or cell division

• Hormones, neurotransmitters, environmental change result in changes within the cell itself that are mediated by signal transduction (cell signaling).

• How does a hormone on the outside change the inside?

How is the need for a change in cell function communicated intracellularly?

• Ligand: is a generic term for something (like a hormone) that binds a receptor protein and changes the function on the receptor on the intracellular side.

• Classes of Signals carried between cells:– Electrical: requires gap junctions to pass between cells,

voltage gated channels to pass along length of cell (singular)– Endocrine: requires release of hormone (specific ligands)

that are carried by blood to target– Paracrine: requires release of hormone (specific ligands) that

are carried only to nearby cells (not blood)– Autocrine: ligand is released and finds receptors on the

same cell that released it.

The beauty of the receptor-ligand interaction.?• Many ligands are unable to cross the lipid bilayer of the plasma

membrane, and cannot enter the cytosol due to their size or polarity.

• Protein receptors can have very precise specificity for a classes of ligand or even sub-classes.

• Target cells can express no receptors, just one type of receptor, or many different types of receptor for the same or different ligands.

• The number and activity of the receptors on the plasma membrane can be modified to reflect the cells needs/status.

• Biggest Ligand Limit: how do you ensure that enough ligand is released so that enough reaches the target cell at the right time and how expensive will it be to produce or release enough ligand to create change?

Hormones are often present at mico or nanogram/liter quantities in the blood. Amplification creates massive effects in the target cell!

Steroids and thyroxine are lipid soluble hormones requiring carriers for transport in the blood, but they can freely diffuse THROUGH the plasma membrane . Once inside they find new receptors that carry then to specific “Hormone Response Elements” on the DNA modifying DNA transcription, thus changing production of things like different receptor classes/subclasses that can change future responsiveness.

Receptor “isoforms” are receptor proteins with a similar but not identical amino acid sequence. Receptor isoforms are suggestive of a common genetic heritage and can have similar or dissimilar enzymatic activity outcomes in cells.

Consider the isoforms of the histamine receptor and its importance for letting different pharmacological mimics of histidine control GERD and another to control your allergies.

Diphenydramine-BenadrylLoratidine-Claritin

Cimetidine-Tagamet

Possible sleep disorder use

Possible Asthma/Inflammatory useKatzung, 2007

The serotonin family of ligand isoforms is a classic with diversity that evolved to permit us to create specific tailored responses to a series of receptor isoforms that can create a myraid of potential tailored cell responses.

Katzung 2007

Signal amplification can be tied to a myraid of different reactions just inside the cell, there are four classics however that predominate. We call these second messenger systems, although some may be a third or even fourth order product resulting from the original ligand-receptor binding on the plasma membrane.

Classic Second Messenger Methods:• G-proteins: release of inhibitor or stimulatory alpha-subunit• Adenyl cyclasecAMP• Guanyl cyclase cGMP• Phospholipase C DAG + IP3• Entry of calcium into the cytosolCa++ dependent change in

enzyme activity may occur.• Activation or deactivation of Phosphokinase(s)/Phosphatase(s).• You also need to have a way to “reverse” the signal cascade!

The number of receptors, G-proteins, types of alpha subunit and amount of phosphodiesterase can all be used to regulate cellular responsiveness.

Cholera toxin blocks G-a-s degradation.

Pertussis toxin blocksG-a-I activation

Epinephrine and norepinephrine have the ability to modify the ability of CD4+ B-cells so they produce more IgG antibodies by increasing cAMP. (Kin and Sanders, 2006)

cAMP inhibits T-cell activation through a PKA type I–Csk–Lck inhibitory pathway. In T-cells, cAMP inhibits TCR-induced T-cell activation and thereby exerts important immunoregulatory functions through a receptor–G-protein–AC–cAMP–PKA type I–Csk inhibitory pathway assembled in T-cell lipid rafts and acting on the Src-family kinase Lck (EP-R, prostaglandin E2 receptor). Taske and Stokka. The molecular machinery for cAMP-dependent immunomodulation in T-cells. Biochemical Society Transactions (2006: 34)

The PL-C product DAG is an important activator of PK-C, and PK-C is significant for the function of the immune system and dysfunctions related to cancer/tumor suppression.

The activity of cGMP or cAMP is often regulated indirectly by stimulating or inhibiting phosphodiesterase that destroy the original signal.

There are two specific types of phosphodiesterase:1) cAMP + H2O AMP + H+

Inhibited by theophylline or caffeine

2) cGMP + H2O GMP + H+Many important inhibitorsViagra is a classic example

Guanylyl cyclases can be a membrane-ligand/receptor form or a soluble cytosolic form.• cGMP has a less global role than cAMP, but is no less critical for our

survival!• Guanyly cyclase has homology with adenylyl cyclaseTHERE ARE TWO FORMS OF GUANYLYL CYCLASE:• 1) The classic soluble guanylyl cyclase is activated by nitric oxide

(NO). NO binds the haem at the center of the enzyme and stimulates cGMP production.

• 2) Membrane-Receptor-Ligand may be important for the function of atria natiuretic peptide and the promotion of Na+ diuresis in the kidney when your blood pressure is too high.

• cGMP Roles:– Regulation of cGMP gated ion channels in retina– Regulation of protein kinase G (PKG)– Regulation of cGMP dependent phosphodiesterases

How does cGMP mediate vasodilation? Viagra inhibits cGMP phosphodiestrase resulting in excessive activation of protein kinase G (PKG). We will see this when we travel to Gunderson-Lutheran in the form of a nitro pill given to persons who experience angina prior to or during an angioplasty.

Receptor Tyrosine Kinases (RTK) represent a classic and simple way to mediate signal transduction.Proteins encoding RTKs contain four major domains (regions): • An extracellular ligand binding domain what binds here? • An intracellular tyrosine kinase domain what happens here?• An intracellular regulatory domain what binds here?• A transmembrane domain what type of amino acids would you expect here?

• In short: RTKs permit the receptor to remove a phosphate from ATP and attach it to a specific tyrosine on the target molecule.

• In short: Some RTKs autophosphorylate themselves.

• In short: the highly electronegative phosphate alters the conformation of the target protein and changes its function.

• Evolutionarily: the amino acid sequences of the tyrosine kinase domains of RTKs are highly conserved with those of cAMP-dependent protein kinase (PKA) within the ATP binding and substrate binding regions.

• Based upon the presence of these various extracellular domains the RTKs have been sub-divided into at least 14 different families, seven classic (common) classes are as follows:

RTKs are monomers that “may” form dimers in the plasma membrane.Amino Acid Consensus Sequence on RTK Target peptide: -(Lys/Arg)-X-X-(Asp/Glu)-X-X-X-Tyr- or -(Lys/Arg)-X-X-(Asp/Glu)-X-X-Tyr-

Many types exist that differ mostly with respect to what is located on the extracellular side, whether the “form” a dimer and the number of cytosolic kinase domains.

These RTK actions determine normal cell growth, apoptosis and movement of glucose transporters to the plasma membrane.

How does interferon moderate cell function by changing transcription with a tyrosine kinase? (DeFranco, Locksley and Roberston 2007)

Protein Kinase C (PKC): composed of a family of at least 10 isomers, generally bound to plasma membrane

PKC Activation typically linked to DAG (diacylglycerol)

Some isoform activity regulated by Ca++ and Zn++

Target sequence for phosphorylation: -Ser/Thr-X-Arg/Lys-

Activation of PKC may also modify Ca++ permeability

Ca++/Calmodulin Dependent Protein Kinase

This is a big-league player! Increased Ca++ is the signalCalmodulin looks like a dumb bell and when 4 Ca++ are attached

it moves ONTO its target proteinsATTACHMENT of Calmodulin means Ca++ is present and kinase

activity is initiated.Capability of autophosphorylation and targets with Ser/Thr rich

amino ends of pertide chainsKinase itself has three domains: catalytic-regulatory-associationClassic targets: myosin light chain kinase and transcription

factors

What goes up must come down:The role of phosphatases

• Phosphatases remove phosphates from specific targets and therefore reverse the signals generated by kinases.

• Specific for Targets:Tyrosine-PO4 or Serine/Threonine-PO4

• Activity tends to be constitual (not regulated), however there are a few VERY important exceptions to this rule….i.e. glycogen phosphorylase

How do all these signal systems work together to let an antigen presenting dendritic cell cause a T-cell to change its function? (Part #1) (DeFranco, Locksley and Robertson 2007)

Green=MHC

Purple: receptor for Antigen presentign cell detects problem

How do phospholipase-C, IP3 , calcium, phosphatases, and protein kinase D mediate this effect fo change transcription in the T-cell? (Part #2) (DeFranco, Locksley and Robertson 2007)