PHGY 170 Mod 4 - Cell Communication & Cell Death

Extracellular Communication

when a signal is received from outside the cell

Intracellular Communication

external signals cause changes within a cell

Direct Cell-to-Cell communication: Gap Junctions

• cells that are touching can communicate using gap junctions

• gap junctions are made of connexons which dock together to form channels from one cell to another

• allows chemical signals to move directly between cells

Specifics of Gap Junctions

• not all biomolecules can pass through gap junctions

• only small particles such as ions and small signalling molecules can pass, while larger molecules such as proteins and carbs cannot

• excitable cells like cardiac muscle can pass electrical signals as well as chemical signals through gap junctions

• gap junctions are not open doors that allow a constant free exchange of signals

• they are highly regulated and can open and close as appropriate

  • this gating is a defence mechanism so a cell can protect itself if something dangerous is happening in a neighbouring cell

cell to cell communication: secretions

• cells that are not touching can communicate through secretions

Autocrine secretions → substances are released and have an effect on the same cell

Paracrine secretions→ substances are released and have an effect on nearby cells

Endocrine secretions → substances are released and have an effect on distant cells

Neurotransmitters → substances are released by a nerve terminal into the synapse

Secretions: neurotransmitters

• synaptic secretions occur where a nerve cell axon terminates on a target cell

• when an excitatory signal comes down the axon to the synapse, neurotransmitters are released into the synapse where they either bind to a receptor on the target cell, are degraded by enzymes in the synapse, or are taken back up by the nerve cell

• regardless of their fate, the presence of neurotransmitters in the synapse is very transient

components of the signal transduction pathway

signal → can be either membrane permeable or membrane impermeable

receptors → the receptors interact with the signal

signalling proteins → signalling proteins help conduct the signal intracellularly

second messengers → non-protein molecules that help conduct the signal intracellularly

Structure of a signalling pathway

Membrane Permeable Signal Molecule→ molecules bind to receptor proteins in the cytosol

Membrane Impermeable Signal Molecule → binds to transmembrane cell surface receptor proteins which activate second messengers

Signalling Proteins and Second Messengers → amplify, process, and distribute incoming signals from both classes of signal receptor proteins

Cytoplasmic Effectors → some signals are sent to effector proteins in the cytosol. this is typically a fast, short lived response to the activation of a signalling pathway

Nuclear Effectors → some signalling pathways terminate at effectors in the nucleus. these effectors are transcription factors that control gene expression. this results in a slower, more prolonged response to a signalling pathway

Linear signal transduction

• one receptor interacts with one signalling protein or second messenger

convergent signal transduction

• several receptors share common signalling proteins or second messengers

divergent signal transduction

• a single receptor can interact with multiple signalling proteins or second messengers

multi-branched signal transduction

• a combination of convergence and divergence may be happening at the same time

membrane impermeable ligands

• the majority of signal molecules are membrane impermeable

• ligands that cannot penetrate the membrane bind to receptor proteins on the cell surface

• cell surface receptors can be grouped into 5 classes based on structure, binding partners, and cellular location

membrane permeable ligands

• membrane permeable signal transduction molecules are mainly steroids

• ligands that are able to penetrate the membrane are not limited to membrane receptors and can interact with cytosolic receptors

physical signals

• physical signals like pressure, temperature, and light can trigger the signal transduction pathway instead of ligands

receptors (overview)

• receptors are often found on the plasma membrane but can also be found in the cytoplasm of a cell

  • g protein coupled receptors (GPCR)

  • ion channels

  • guanylate cyclase

  • protein kinase receptors

  • transmembrane scaffolds

  • nuclear receptors

• the 6 classes of receptors detect an array of environmental stimuli

G protein coupled receptors

• GPCRs represent a superfamily of receptors with hundreds of genes coding for different receptors

• involved in many functions such as smell detection, and fight or flight activation

structure

• a combination of seven transmembrane domains (H1 to H7) as well as heterotrimeric G protein with aplha, beta, and gamma subunits that interact with each other

function

• the binding of a ligand to a GPCR causes a conformational shape change in the receptor that leads to the activation of the coupled G protein subunits

Ion Channel Receptors

• aka ligand gated channels

• a type of channel that can exist in the plasma membrane

• transmit signal information by permitting ions to flow from one side of the membrane to the other

• when their specific ligands bind, the channels udnergo a conformational change in shape that opens their pores and allows the ions to flow through

• unlike other receptor types, these proteins are not enzymes

• ion channels are responsible for voluntary muscle contraction

• this type of signalling is common for much of the communication between nerve cells through the release of neurotransmitters

Guanylate Cyclase Receptors

• a receptor that can be found both bound to the membrane and soluble within the cytosol

Structure

• membrane bound guanlyate cyclase contains an externalized ligand binding domain, a transmembrane domain, and an internal catalytic domain

• the soluble form of guanylate cyclase serves as a target for some membrane soluble ligands in addition to mediating some intracellular processes

Function

• when the catalytic receptor is activated, the catalytic domain of membrane bound guanylate cyclase converts guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP)

• cGMP then binds to other signalling proteins to initiate cellular processes

• guanylate cyclases play an important role in vision as they help convert a light signal into an electrical signal in the eye

Protien Kinase Receptors

• human cells express hundreds of different protein kinases

• not all protein kinases are cell surface receptors, many are cytosolic proteins that also participate in signal transduction, alter enzyme activity, or other cellular processes

• the action of protein kinases is to phosphorylate other proteins that contain serine, threonine, or tyrosine residues

• protein kinase receptors are especially important in clinical settings because their dysfunction is associated with the development of a number of cancers

• there are 2 classes of protein kinase receptors:

  • receptor tyrosine kinases (RTK)

  • serine/threonine kinases receptors (S/TRK

Protein Tyrosine Kinase Receptor Ligand Binding

Inactive → before ligand binding the inactive receptors are separate polypeptides with inactive tyrosine domains

Dimerization → binding to a signalling molecule causes the two subunits of the receptor to join together or “dimerize,” forming a dimer. once dimerized, the kinase is active

Transautophosphorylation → transautophosphorylation occurs when the cytoplasmic tail of one subunit is brought close to the tyrosine kinase domain of the other subunit, and the opposite domain is phosphorylated on specific tyrosine amino acids

Binding Sites → the resulting phosphotyrosine amino acids are binding sites for additional signalling proteins that pass the signal along the pathway

Resetting → the ligand is released, and the amino acids are dephosphorylated by phosphoprotein phosphotases. the kinase resets itself to its inactive state of two separate polypeptides

Transmembrane Scaffold Receptors

• transmembrane scaffolds are different from other membrane receptors in that they do not always have a distinct single function

• this type of receptor tends to form in large clusters of receptors abd signalling proteins with complex interactions

• by doing this, they can regulate signal transduction

• the scaffold proteins themselves determine which signalling proteins can ind to a complex, associating with the membrane receptor, and form what is called a signalling scaffold

Functions

• bring signalling proteins together

• regulate signal transduction

• localize signalling proteins to specific cellular areas

• isolate specific signalling pathways

Nuclear Receptors

• nuclear receptors are receptor proteins that are found in the cytosol of cells

• certain ligands, such as steroids, can freely cross plasma membranes and bind to these intracellular receptors

• once bound, these receptors move through nuclear pore complexes directly into the nucleus

• once inside the nucleus, the activated receptor complex can bind to a specific DNA sequences called steroid response elements (SREs) to control the expression of genes

• since they help to regulate gene expression, this class of receptors are also called transcription factors\these types of receptors also play a role in response to toxic substances

Signalling Proteins

• the primary purpose of signalling proteins is to transmit and amplify signal information

• signal proteins can also mobilize second messengers, which are non protein molecules that can ling signalling proteins together into further signalling pathways or have direct actions on their own

mobility → signalling proteins are highly mobile and can diffuse rapidly through the cytosol. if membrane associated, they move rapidly within the plasma membrane

catalysis → signalling proteins are either enzymes that can catalyze chemical reactions for signal amplification or they are capable of binding to enzymes

Signalling Proteins: G proteins

• G proteins are proteins that bind to GTP and propagate signals

Monomeric G proteins

• monomeric G proteins are single polypeptides that contain at least 2 different binding sites (one for GTP or GDP and one for the target protein) and a GTPase domain

• they are NOT coupled to GPCRs

• when GTP is bound, it is in an activated state and can bind to its target protein

• the GTPase can then cleave the GTP to form GDP

• eventually the GDP is released and GTP can then bind again to reactivate it

Heterotrimeric G proteins

• similar in function to monomeric G proteins except they contain 3 different polypeptides

• these G proteins are anchored to the plasma membrane and are activated by the G protein coupled receptors already mentioned

• the alpha subunit is analogous to the monomeric G protein in that it binds GTP/GDP and a target protein

• the beta/gamma subunits are attached together and their primary function is to stabilize the inactive (GDP bound) form of the alpha subunit

Activity of G proteins: Binding

• the heterotrimer containing the alpha and beta/gamma subunits is bound to GTP. this is the inactive form

• when a ligand binds to the receptor, it changes conformation to interact with the heterotrimeric G protein

Activity of G proteins: Separation

• the receptor protein causes exchange of GDP with GTP on the alpha subunit

• the heterotrimer separates into separate alpha and beta/gamma subunits

• the G proteins are active

Activity of G proteins: Propagate

• while separated, the alpha and beta/gamma subunits bind downstream targets, propagating the signal pathway

Activity of G proteins: cleave and reform

• the alpha subunit cleaves GTP to form GDP, alpha and beta/gamma subunits bind to reform the heterotrimer

• this returns the heterotrimeric G protein complex to the inactive form

signalling proteins: protein kinases

• protein kinases are enzymes that attach phosphate groups to tyrosine, serine, and theronine

• in addition to receptor protein kinases, there are non receptor protein kinases

• the majority of protein kinases are non receptor, cytosolic signalling proteins

• cytosolic protein kinases can act as intermediates, in that once they are activated they can activate other protein kinases, other signalling proteins, or they can directly phosphorylate effector proteins like enzymes

• in general, phosphorylation of target proteins leads to their activation byt some proteins are inactivated by phosphorylation

• some protein kinases can enter the nucleus but they do not interact with DNA directly

• instead, they can phosphorylate proteins that do interact directly with the DNA

Signalling proteins: calcium binding proteins

• Ca2+ is an ion in the cell that has a number of function

• typically, intracellular calcium is kept at low concentrations so when levels increase due to a signalling event, it can interact with certain proteins causing downstream effects

• an example of calcium binding protein is calmodulin

• when calcium concentrations rise, calcium binds to calmodulin inducing a conformational change that allows calmodulin to bind to its target protein

Adenylyl cyclase

• another major class of intracellulr signalling proteins is adenylyl cyclase

• they are related to guanylyl cyclase in that a nucleotide triphosphate is converted into another form

• ATP is converted into cAMP, perpetuating the signal

• in contrast, adenylyl cyclase is not linked to membrane receptors

• instead, adenylyl cyclase binds to the alpha subunit of the heterotrimeric G proteins, which is why it is designated as a signalling protein instead of a receptor type

Signalling proteins: adenylyl cyclase subunits

• there are two types of heterotrimeric G protein alpha subunits:

  • alpha s stimulates adenylyl cyclase

  • alpha i inhibits it

• these two different forms of alpha subunits form parts of different heterotrimeric G proteins and are linked to different GPCRs which highlights a level of cellular decision making in which multiple pathways converge to allow a single response

Signalling proteins: lipid kinases

• lipid kinases are the class of signalling proteins that phosphorylate phospholipids in the cytoplasmic leaflet of the membrane

• in general, lipid kinases add a phosphate to the polar head group

• phosphorylation of the polar head group results in a conformational change in the phospholipid and allows it to bind to its target protein in the membrane to pass the signal down the pathway

• some phospholipids can be phosphorylated more than once to become an active signalling molecule

signalling proteins: adaptor proteins

• nearly all signal transduction pathways have another class of proteins that are neither receptors or enzymes

• these are known as adaptors

• these proteins have different domains that recognize phosphorylated amino acids or other activated structures on signalling proteins

• these domains along with others form the glue to hold elements of signalling networks together at the right time and place in a cell

• the adaptor proteins are important to allow cascades to be associated in the right space and time to fulfill their tasks when and where they are needed in the cell

features of second messengers

key features

• small in size

• diffuse rapidly in the cytosol or membrane

• can amplify signals so that the interaction of just a few ligands with their receptors can trigger a much larger response within a cell by mobilizing second messengers

• they do not hang around in the cytosol for too long

• second messengers such as cAMP and cGMP are degraded by specific enzymes called phosphodiesterases, while ionic messengers such as Ca2+ are sequestered into cellular organelles

• other examples include hydrophobic molecules such as diacyglycerol (DAG) and inositol triphosphate (IP3) and some gasses like nitric oxide (NO)

summary of signalling pathways

heterotrimeric G protein signalling cascade

1. the signal transduction is initiated by the binding of a ligand to the GPCR. binding of the receptor allows the receptor protein to interact with the heterodimeric G protein

2. the ligand bound receptor stimulates the replacement of GDP for GTP in the alpha subunit. this causes the heterodimeric G protein to disassociate from the receptor and itself to leave a G (beta and gamma) subunit and an activated G alpha s GTP. the G apha s GTP then binds and activates the signalling protein adenylyl cyclase to convert ATP to cAMP, a second messenger

3. next, cAMP can bind to another signalling protein, protein kinase A (PKA). inactive PKA is a tetrameric protein with two regulatory subunits. the binding of cAMP to the regulatory subunits causes the protein to dissociate and release the active catalytic subunit. once active, the catalytic subunit can phosphorylate a number of cellular proteins

4. active PKA catalytic domains can enter the nucleus. a common nuclear target is the cyclic AMP response element binding protein (CREB). once phosphorylated by PKA, CREB binds CBP (CREB binding protein) and together, the two proteins can interact with DNA to initiate transcription

Summary

1. GPCRs

2. cAMP

3. PKA

4. CREB

Phospholipid Kinase Signalling Cascade

1. the signal transduction pathway is initiated by the binding of a ligand to the GPCR. binding of the receptor allows the receptor to interact with the heterotrimeric G protein. the ligand bound receptor stimulates the replacement of GDP for GTP in the G alpha subunit. this causes heterotrimeric G protein to to dissociate from the receptor and itself to leave a G beta gamma subunit and an activated G alpha GTP

2. the G alpha GTP binds the phospholipid kinase signalling protein phospholipase C (PLC)

3. an activated PLC breaks down the membrane phospholipid phosphatidylinositol 5,5-biphosphate (PIP2) to release two second messengers: DAG and IP3

4. IP3 diffuses freely in the cytosol and activates its receptor on the ER, which opens a ligand gated calcium channel. Ca2+ leaves the ER and, acting as a second messenger, can activate a number of calcium binding proteins

5. together, the membrane bound diacylglycerol and cytosolic Ca2+ bind to protein kinase C (PKC), resulting in its activation. activated PKC has numerous cellular targets it can phosphorylate to modulate the target’s activity

Summary

1. GPCR

2. PLC

3. PIP2/IP3

4. Ca2+

5. PKC

protein kinase signalling cascade

1. fibroblast growth facgtors (FGFs) are a class of proteins that stimulate the growth of most mammalian cells. FGFs bind to a family of receptor proteins called FGF receptors (FGFRs). FGFR is a homodimeric receptor kinase (tyrosine kinase). binding of FGF to FGFR causes the subunits to dimerize. once bound together, the FGFR undergose tyrosine transautophosphorylation to form phosphotyrosines on the cytoplasmic side. these phosphotyrosines can be bound by many different proteins

2. one such binding protein is the adaptor protein Grb2. binding to a phosphotyrosine causes Grb2 to undergo a conformation change to bind to Sos. Sos activation leads to its binding to a monomeric G protein Ras. Binding of Sos to Ras replaces the GDP with GTP and the now active Ras can bind to a serine/theronine kinase called Raf. Activated Raf can phosphorylate another protein kinase called MEK, which will in turn phosphorylate another serine/threonine kinase called Erk

3. phosphorylated Erk forms a dimer and can phosphorylate other signalling proteins in either the cytosol or the nucleus. Erk enters the nucleus to activate transcription factors, ultimately initiating transcription

Summary

1. FGFs

2. Grb2 & intermediates

3. Erk

lysosomes, proteosomes, peroxisomes

lysosomes → organelles that break down misfolded and damaged organelles, nucleic acids, lipids, and more

proteasomes → protein complexes that specifically break down damaged and misfolded proteins in the nucleus and cytosol

peroxisomes → peroxisomes handle dangerous free radicals including reactive oxygen species. thes are also problematic to the cell and needs a safe place to use these chemicals

getting cargo to the lysosome

• misfolded or non functional proteins ad other cellular contents are tagged for delivery to the lysosome in an endosome via the endomembrane system

• cargo is targeted to the lysosome by a specific mannose 6 phosphate (M6P) sugar tag

• the enzymes that degrade these damaged proteins are also directed to the lysosome with an M6P tag

vesicles

• the engulfed proteins including the membrane proteins and suluble proteins are delivered by vesicles that empty their contents by fusing with the lysosome and are digested by the proteases

Proteases

• the proteases are synthesized in the ER, tagged with M6P, and delivered to the lysosomes by vesicles

• they digest both soluble proteins and membrane proteins in the lysosome

digestion in the lysosome

• lysosomes are mainly responsible for the breakdown of proteins that are not endogenous to the cell or from other organelles

• the lysosome contains high concentrations of proteases, which cleave both membrane proteins and proteins contained within the lysosome

• the lysosome also contains enzymes that cleave and digest fats and sugars and can even engulf other organelles like damaged mitochondria or bacteria

• once large molecules have been broken down into their basic parts like proteins into amino acids they are transported to the cytosol so the cell can reuse them

protein degradation by the proteasome

cytosolic proteins

• cytosolic proteins that have been misfolded or damaged are tagged with a polyubiquitin chain, which is composed of multiple molecules of ubiquitin

• multiple ubiquitins are required for the protein to be targeted and recognized by the proteasome and degraded

nuclear proteins

• proteasomes are also located in the nucleus so the cell can degrade unwanted nuclear proteins without having to export them to the cytosol

• damaged histones, for example, can by polyubiquitinated in the nucleus and degraded by nuclear proteasomes

function of peroxisomes

• oxidizing agents like peroxides, ions, and free radicals are very hazardous to the cell

• peroxisomes serve as a place to keep and use these reactive oxygen species safely using enzymes including catalase

• peroxisomes are small, membrane enclosed organelles and contain enzymes that catalyze a variety of metabolic reactions

• essential peroxisome proteins are called peroxins, they are synthesized in the cytosol and are targeted to the peroxisome by specific peroxisomal targeting signals (PTSs)

• although they are hazardous, peroxisomes also carry out important decomposing functions for some cargo such as uric acid, amino acids, and long chain fatty acids

Apoptosis

• programmed cell death

• an energy consuming process

• used to protect the body from damaged cells that no longer function properly

• also used in development, ie. to remove the webbing from between fingers and toes in fetal development

• kaboom! (but slowly, neatly. very demure, very mindful)

mechanisms of apoptosis: initiation

• apoptosis is initiated by two different pathways: intrinsic and extrinsic

• the cell initiates apoptosis itself

• the intrinsic pathway originates in the outer membrane of the mitochondria

• intracellular signals such as sever DNA damage, ROS, toxins, or other trauma will turn on the intrinsic pathway in the cell

• extrinsic signals initiate apoptosis in the cell

• the extrinsic pathway uses a plasma membrane receptor called the death receptor

• neighbouring cells such as immune cells will release death ligands which bind to the death receptor on a damaged cell which activates additional signals that lead to apoptosis

mechanisms of apoptosis: membrane blebbing and enzyme activation

• the cell begins to shrink and form blebs (small protrusions from the membrane)

• this is the first visible signal that a cell is undergoing apoptosis

• enzymes termed caspases are activated

• the initiator caspases are activated by either the extrinsic pathway or intrinsic pathway

• these caspases will cleave and activate other caspases called executioner caspases

mechanisms of apoptosis: cell structure changes

• after the executioner caspases are activated the cell changes structure

• DNA is fragmented, often between histones, and DNA repair halts

• the nuclear membrane breaks down and the nucleus disappears

• the cytoskeleton is disassembled and the plasma membrane phsopholipid content changes with scramblases, woth PS (phosphotidyl serine) being exposed on the exoplasmic leaflet of the plasma membrane

• organelles persist, and are enclosed in apoptotic bodies

mechanisms of apoptosis: engulfment

• phagocytes endocytose the apoptotic bodies to dispose of them

• these are then safely digested by the phagocytes lysosomes

• this causes a minimal amount of disturbance to the cells and surrounding tissues

necrosis

• resulting from cellular injury that cannot be repaired

• the major pathway of cell death as a result of damage that cannot be repaired. the cell’s organelles are not able to function and it dies

mechanisms of necrosis: damage

the cell is damaged beyond repair. there can be many causes

toxins → ie. bacteria, drugs, chemicals

extreme heat or radiation → denatures proteins, damages DNA

freezing → ice crystals puncture the cell membranes and organelles

ischemia → blood flow is stopped to the tissue; lack of oxygen, glucose, etc, prevents the cell from receiving life essentials

pathogens → bacterial or fungal infections

mechanical trauma → physical injury to the cell

mechanisms of necrosis: swelling

• the organelles begin to lose their structures and swell

• vacuoles, or undefined bodies, form in the cell

• depending on the type of damage, the DNA may be degraded

mechanisms of necrosis: destruction

• the cell membrane and remaining organelles lose structural integrity

• holes can be observed using microscopy

• the cellular contents spill out of the cell, producing inflammatory signals

• the mitochondria’s proteins are released and lysosomal contents are exposed

• cells nearby are exposed to these remains of the cell, and are also damaged or have apoptosis signalling triggered

• unlike apoptosis, it is difficult for the body to clean up the cellular remains after necrosis

Apoptosis vs Necrosis