Cell Biology- Chapter 16

Principles of Cell Signaling

Signal transduction is the conversion of information to different forms

Cells can communicate across variable distances

• Paracrine Signaling:

In paracrine signaling, cells release signaling molecules that affect nearby target cells. The signaling molecules, often called local mediators, diffuse through the extracellular fluid to reach their targets. This type of signaling is important in processes like inflammation and tissue repair.

• Neuronal Signaling:

Neuronal signaling, also known as synaptic signaling, involves the transmission of signals between neurons or between neurons and other types of cells (like muscle cells). This occurs through synapses, where neurotransmitters are released from the presynaptic neuron and bind to receptors on the postsynaptic cell, leading to a response.

• Contact-Dependent Signaling:

In contact-dependent signaling, the signaling molecule is bound to the surface of the signaling cell, and it interacts directly with receptor molecules on the surface of the target cell. This type of signaling requires direct cell-to-cell contact and is crucial during development and immune responses.

• Endocrine Signaling:

Endocrine signaling involves the release of hormones into the bloodstream by endocrine cells. These hormones travel through the blood to reach distant target cells throughout the body. This type of signaling is essential for regulating various physiological processes, such as metabolism, growth, and reproduction.

Receptor location in a cell depends on the chemical nature of the signal molecule

• cell surface receptors are located on the plasma membrane- signal molecules that are polar require surface receptors, and activation initiates intracellular signaling that drives responses

• Intracellular receptors are located within the cell- ligands are hydrophobic and can diffuse across membranes. mostly act as ligand-dependent transcription factors

A single signal molecule can produce varying effects based on receptor and cell

• acetylcholine is a polar example

Integration of different signals results in different cellular responses

• not uniform

• signaling from different molecules can integrate to produce different effects

• absence causes apoptosis- anoikis (homeless) cells result when an epithelial cell is detached from the lamina.

A single signal can produce fast and slow effects

A single signaling pathway can branch

• During signal transduction, the pathway can branch out, meaning the signal is relayed to multiple downstream targets. This branching allows the cell to coordinate a variety of responses simultaneously. For example, one branch might lead to changes in gene expression, while another might alter cell metabolism or cytoskeletal organization

Pathways can also converge

• Convergence occurs when different signaling molecules or pathways lead to the activation of the same intracellular signaling molecule or pathway. For example, different growth factors might activate the same kinase, leading to cell proliferation.

Signal pathways can be modulated by positive and negative feedback

• positive: downstream factor stimulates activity of an upstream factor

• negative: downstream factor inhibits activity of an upstream factor- can result in an oscillatory response

Common molecular switches: phosphorylation and GTP binding

• Kinase and phosphatase: signaling by protein phosphorylation

• GTP binding and hydrolysis: signaling by GTP binding proteins

Monomeric (small) GTPases are controlled by GAPs and GEFs

• GEF- guanine nucleotide exchange factor

• GAP- GTPase activating protein

• G proteins function does not depend on the energy of GTP hydrolysis, GTP acts as a switch, and non-hydrolysable forms of GTP turns G proteins on permanently

• RAS is a G protein in pro-survival pathways, mutation in its GTPase domain leads to cancer

General receptor types- ion channel coupled receptors

• ion channel coupled receptors open (or close) upon binding of ligand to extracellular or intracellular domains

• Neurotransmitter receptors that are ion channels are said to mediate ionotropic signaling. such signaling directly alters membrane potential.

General receptor types- G protein coupled receptors

• GPCRs are associated with inactive trimeric G protein complex

• the associated proteins are distinct from monomeric G proteins

• Ligand binding releases the subunits of the associated G protein, which each mediate different signaling branches

General receptor types- Enzyme coupled receptors

• many enzyme-coupled receptors have intrinsic catalytic domains that are activated upon ligand induced dimerization- some lack catalytic function, instead recruiting enzymes

• many ECRs have kinase functions that promote signaling complex assembly through site specific phosphorylation

• receptor tyrosine kinases and receptor serine-threonine kinases are common

G Protein-Coupled Receptors

GPCRs have seven membrane-spanning alpha helices

• GPCRs are synthesized as 7-pass integral membrane proteins

• GPCRs are called serpentine receptors

Ligand binding to a GPCR activates the associated G protein

• GPCRs are named because their function depends on an association with G proteins- trimeric G protein is lipid-linked to the plasma membrane

• In the absence of ligands, the receptor has little affinity for the G protein inner membrane

• Ligand binding induces a conformation change in the alpha subunit of the complex

• The GTP bound alpha subunit splits away from the beta and gamma complex and each passes the signal along to their down downstream targets.

GTP hydrolysis inactivates the alpha subunit and reassembles the G protein complex

• activated alpha subunit activates a target protein

• The alpha subunit as intrinsic GTPase activity and in time, will hydrolyze its bound GTP to GDP

• Once bound to GDP, the alpha subunit loses its affinity for the target protein and rebinds the beta-gamma complex

• the beta-gamma complex prevents release of GDP, and the G protein returns to an inactive state.

• The beta-gamma complex acts as another type of G protein regulator- the guanine nucleotide disassociation inhibitor (GDI).

Alpha and beta-gamma G protein subunits have distinct functions

• mutually inhibit each other when bound together

• Upon separation, alpha and beta-gamma can interact with their own target proteins

• Neuronal signaling the opens an ion channel directly via intracellular signaling is metabotropic

• Ionotropic signaling opens channels directly

• adenylyl cyclase partners with the alpha subunit.

Alpha subunits signal through second messengers

• alpha subunits signal through their association with enzymes

• the reaction catalyzed by the enzyme (promotion or inhibition) depends on the particular alpha subunit

• Signal molecules produced (or released) via enzymes (or transporters) are referred to as second messengers, which amplify and pass forward a signal

• Cyclic AMP, inositol triphosphate, diacylglycerol, and Ca++ are common second messengers

• a single activated enzyme can produce many secondary messengers

Gas and Gai subunits alter cyclic AMP (cAMP) concentrations

• cAMP is a metabolite of ATP

• Adenylyl cyclase catalyzes phosphodiester bond formation between a 5’ phosphate and 3’ hydroxyl of the same ribose

• Gas activates AC, Gai inhibits it

cAMP is an activator of protein kinase a (PKA)

• cAMP had multiple targets that vary to cell type- cAMP gated/cAMP sensitive

• Protein Kinase A depends on cAMP binding for full activity

• Once bound to cAMP, PKA phosphorylates its protein targets using ATP as a phosphate donor

• PKA can initiate a phosphorylation cascade, where each protein target is a kinase that phosphorylates the next kinase in the sequence

PKA can also phosphorylate targets in the nucleus

• Phosphorylation of transcriptional regulators by PKA is one example (of many) signal transduction that alters gene expression

• Think about the roles of PKA in the cytosol and the nucleus. Do you think PKA has a nuclear localization sequence? Why or why not?

Activation of different GPCRs controls heart rate

• two different types control heart rate: Parasympathetic (releases acetylcholine) and Sympathetic (releases norepinephrine)

• Cardiac muscle cells have GPCRs that modulate cAMP concentration

• in cardiac myocytes, cAMP promotes Ca++ influx, which enhances rate of contraction

Cholera and pertussis toxins increase cAMP through different mechanisms

• toxins can modify alpha subunits with ADP

• Cholera enhances cAMP signaling in enterocytes by locking Gas in an active state, leading to the increased expression of the chloride channel CFTR on the apical membrane, causing water secretion (diarrhea)

• Pertussis toxin enhances cAMP signaling in immune cells by preventing activation in Gai- this is hypothesized to inactivate the immune cells

Gq signals through phospholipase C (PLC) and protein kinase C (PKC)

• G (alpha-q) activates phospholiase C (PLC), an enzyme that hydrolyzes liquid

• The main substrate for PLC is a membrane glycolipid called phosphatidylinositol 4, 5- biphosphate (PIP2). this yields inositol (IP3) and DAG

• IP3 opens a calcium channel on the ER membrane, calcium and DAG, together activate a protein kinase PKC. it phosphorylates it’s own targets!

• these substances have other functions besides these

Calcium binding regulatory proteins….

• write notes for this on your own time

Gaq signaling in endothelial cells leads to nitric oxide production, which releases smooth muscle cells

• Vasorelaxation can be initiated by several signals, some of them act via endothelial cells that line the inner surface of the vessel

• Acetylcholine (from parasympathetic neurons) activates a Gaq, coupled muscarinic receptor in endothelial cells- the increase in calcium activates the enzyme nitric oxide synthase (NOS)

• Nitric oxide is a gas that freely diffuses into the neighboring smooth muscle cells

• cGMP hyperpolarizes the cell, closing calcium channels and relaxing the cell.

• cGMP is broken down by cGMP phosphodiesterase, the drug sildenafil inhibits cGMP broken down, and prolongs vasorelaxation

Rhodopsin is GPCR activated by light

• Photodetection is an unusual sense in that the sensing cells are depolarized when they are not stimulated.

• Rhodopsin (and opsins generally) is a GPCR the associated with a special alpha subunit called transducin (G alpha-t)

• G alpha-t activates cGMP phosphodiesterase, which degrades cGMP

• falling cGMP levels cause cell hyperpolarization

• because of this, photoreceptors only release neurotransmitter in the dark.

Amplification of GPCR signals produces rapid responses

Enzyme Coupled Receptors

enzyme-coupled receptors either have enzymatic function or recruit proteins that do

• Many enzyme coupled receptors have intrinsic catalytic domains that are activated upon ligand induced dimerization

• Need more notes

Receptor tyrosine kinases autophosphorylate upon ligand-induced dimerization

• RTKs have kinase domains that specify phosphorylated tyrosine residues

• ligands for RKS are typically dimers and upon binding, induce receptor dimerization

• Dimerization brings the kinase domains into close proximity to tyrosine residues; phosphorylation of the receptors creates binding sites for signaling complex completely

• ligand dependent signaling is possible if alterations in regulatory DNA sequences result in high receptor expression

Ras is a monomeric (small) G protein activated by many different RTKs (VERY important for exam!)

• the ras-GEF is a common component of many RTK signaling complexes.

• ras-GEF activates ras, a small G protein (do not confuse with timeric G proteins of GPCRs) by loading it into GTP

• Activated ras passes the signal forward continuously until a Ras-GAP switches it off by promoting hydrolysis of GTP to GDP

Ras initiates a MAP kinase pathway (promotes cell division)

• Mitogen activated protein (MAP) kinase pathways, as the name suggests, are intracellular signaling processes driven by ATP-dependent phosphorylation that promotes cell division

• The general structure: a top-level MAP kinase kinase phosphorylates a MAP kinase kinase, which phosphorylates a MAP kinase, which phosphorylates effector proteins

• Since each activated kinase activates another kinase, this form of signal transduction is a phosphorylation cascade

• no phosphorylation is linear, each kinase has multiple targets

RTK signaling through Akt depends on phosphorylation of PIP2

• the PI3-Kinase/ Akt pathway promotes cell survival - PIP3 recruits a protein kinase that phosphorylates Akt, which was brought to the membrane by another PIP3

• Akt is a serine/threonine kinase (PKB) that passes the signal forward through phosphorylation of its target proteins

• the pathway can be terminated by conversion of PIP3 back to PIP2 by a PTEN enzyme

Akt signaling promotes cell survival through inhibition of apoptosis

• apoptosis is an ATP dependent mechanism

• Bcl2 inhibits apoptosis, is inactive by Bad in the absence of Akt signaling

• activation of Akt results in Bad phosphorylation and release of BCl2

• other regulating proteins include Bim, Bid, etc.

Akt promotes cell growth via activation of Tor

• Akt signaling promotes protein synthesis and increases in cell cize through activation of Tor.

• Rapamycin inhibits Tor function - cells have longer lifespans because of caloric restriction, where reduced Tor activation happens.

Shared targets integrate signaling through GPCRs and RTKs

Protein techniques reveal composition of signaling complexes

• immunoprecipitation uses antibodies to isolate specific proteins from a heterogenous protein mixture

• If proteins interact, they can be co-precipitated

• Co-Immunoprecipitation followed by proteomics can define or confirm composition of receptor-signaling molecule complexes.

Receptor mutants can reveal signaling factor binding sites

• once the components of a signaling complex are known, key amino acid residues required for complex component binding to the receptor can be identified

• Underthaning the molecular basic can be foundational to designing drugs

Experiments with loss-of-function mutants can deduce signal pathway order

Notch signaling is a direct contact-dependent pathway that regulates tissue patterning

• once cells have committed to becoming a neuron, it expresses a Notch ligand called Delta

• Notch signaling suppresses differentiation into neurons, so the lone neuron in a cluster tells all its nearest neighbors to remain as epithelial cells

• In addition to nervous system problems, Notch mutant flies have notches in their wings