BIO315 - Cell Bio

Human Cell Biology

Cell communication

allows organisms to respond to changes in their environment

Intracellular signalling/signal transduction

A signal molecule(ligand) attaches to a receptor which sends a signalling cascade to alter the effector(target protein) and alters the cell behaviour

4 components of cell communication

• Signal molecules

• Receptor proteins

• Intracellular signalling proteins

• target/effector proteins

Extracellular signalling can…

can act over short and long distances

4 Classes of intracellular signalling

contact-dependant, paracrine, endocrine, synaptic

Contact-dependant signalling

cell-to-cell by membrane-bound ligands

Paracrine signal

The ligand secreted by the cell in the ECF to target cells in the near vicinity

Endocrine signal

hormone secreted into the bloodstream and travels to target cell/receptor -> takes longer

Synaptic signal

neurons connected to other neurons/target cells

Receptor Binding types

• cell surface -> on the cell membrane

• intracellular receptors -> within the cell

A combination of biological responses in a cell can…

• have many receptor types and knowing when to respond to a signal depends on the signal type, the concentration of the signal, or the absence of a signal

Cell types and responses

Responses depend on the cell type the signal is given to ex. Acth on heart cells will decrease the firing rate of signal

Signal concentrations are important since…

• Some cells need higher or lower concentrations of a signal to be activated -> varies with the cell

3 classes of cell surface receptors

ion-channel, G-protein, enzyme-coupled

Ion-channel coupled receptor

• transmit by ion-gated channels and a neurotransmitter binds to the channel and causes it to close/open -> excitability of a cell

G-protein receptor

• by heterotrimeric G proteins to regulate membrane-bound target proteins

Enzyme coupled receptor

• receptor has enzymatic activity or uses an enzyme to activate the receptor -> usually has protein kinase activity

Second Messengers

• relay signals from the membrane-bound proteins to effector proteins -> like on/off switches on proteins → relay signals

Importance of intracellular signal proteins

• relay signals, act as a scaffold, spread signal from one pathway to another

Phosphorylation/dephosphorylation

the addition or elimination of a phosphoryl group to a molecule - storage/transfer of free E

Signal by phosphorylation

• Signal in -> protein kinase uses ATP -> signalling protein on ->

Signal by dephosphorylation

• protein phosphatase removes Pi from ATP -> signalling protein off

Kinase cascade

• (multiple protein kinases working - in sequences) one gets activated, and it activates the next, in a signal line -> adds more phosphate groups

Protein kinase vs phosphatase

Protein kinase adds a phosphate while protein phosphatase remove phosphate group

3 Types of kinases

• ser/thr -> phosphorylate the hydroxyl groups in ser/thr

• Tyr -> phosphorylates proteins on tyr 

• dual specificity -> can phosphorylate on all three ser/thr/tyr

GTP/GDP binding signalling

• Causes a change inside the cell from being outside the cell -> on when GTP bound, off when GDP bound

  • When on, they have GTP activity and shut off when hydrolyzing their GTP to GDP

Types of GTP proteins

• heterotrimeric G protein -> help relay signals from G-protein receptors -> many subunits

• monomeric GTPases -> help relay signals from many cell-surface receptors -> one subunit

Regulation of monomeric GTPases

GAP, GEF, and GDI

GAP GTPase

causes the off state by hydrolyzing more GTP - once the G protein is bound to GTP, it will be hydrolyzed to return to the inactive state

GEF GTPase

causes the on state by eliminating GDP and allowing more GTP to bind - release GDP and bond GTP

GDI

binds to G protein in the off-state and prevents the release of GDP -> promotes the off-state

Inhibition steps of signalling

• Most signals have activation and inhibition steps

• Two inhibitory effects in one path can create a positive effect

  • Signal -> protein kinase -> inhibitory protein -> transcription regulator free -> induce gene regulation 

How to ensure signal response and specificity?

High-affinity interactions that are highly specific, Signal threshold, Localization

Localizing by scaffold protein

use of specific binding sites - brings groups of interacting signalling proteins into signalling complexes

Localizing at the site of unactivated receptors

in the scaffold protein that is inactive but proteins are inserted already, they are activated in a downstream manner when the signal protein activates the receptor

Localizing by phospholipids

• docking on the membrane and interacting with other signalling proteins when the receptor on the membrane is activated

  • The head group attracts the proteins to bind t

Importance of interaction domians

• a small region of a protein that has the correct structure to bind to a specific motif of another protein

Induced proximity

signal triggers the assembly of complex proteins by bringing proteins closer together -> triggers the protein to come closer together

interaction domains

-> areas of the proteins where they bind to other proteins

Signal complex in Insulin receptor

Different types of signalling pathway properties

• Response time(synaptic = fast, endocrine = slow)

• sensitivity(receptors, some need lower concentrations of a signal to activate, others higher[C]) -> some extremely sensitive, some need more signal molecule

• dynamic range(adaptation mechanisms, some need specific signals other can have broader ones) - range a pathway reacts to(narrow or broad)

• persistence(+/- feedback) - how long a response lasts(some longer lasting)

• signal processing(ex. Gradual to abrupt) - simple signal to complex response

• integration(many signals can activate one signal)-> need coincidence detectors

• coordination(one signal to a response that activates many signals)

Signal integration/coincidence detectors

• Combinatorial signals -> helped by coincidence detectors

  • Senses when two signals come in at the same time and allow the cell to decide on an appropriate response 

Factors influencing response times

• Depends on what needs to be done -> depends on what the effector protein is and what the end outcome is

Turnover in signalling molecules

• inhibition processes determine how quickly and great the response can be -> activation needs to be quickly revered to make rapid signalling possible

Half-life in signal molecules

• Very stable at steady state, they are built up, if you start low, you will take a lower time to double than if you started higher, and will take longer

• Short-half life is unstable since it can rapidly increase in amount and response time at initiation is very quick

• Short-half life at termination will drop very quickly than a molecule than that of a longer half-life molecule

Signal processing in biological processes

• Some signals need slower responses, like hormones, to be able to fine-tune their signals

• Other systems need faster signalling, like action potentials, to work right -> all-or-one response is maximal

Allosteric Binding

• to make the response more steep, you can incorporate signalling proteins that are controlled by allosteric bonding - very high concentration does this happen 

-/+ feedback

- stops the signal from countiuing

+ continues the signal more and more

Negative feedback

• allows cells to use a small [C] of signals to do what they need

Positive feedback

• gradual increase in [C] that gets more and more, but it will eventually stop -> all-or-none reactions

Regulating signal sensitivity and dynamic range

Adaptation!

Adaptation

the ability of a cell to respond to a wide range of signals [C]

GPCR

G-Protein Coupled Receptor

one ligand can bind to many GPCRs and one GCPR to many ligand types - all are 7 transmembrane and signals through heterotrimeric G proteins

Heterotrimeric G-proteins

all have 3 subunits of alpha, beta, and gamma

• In an unstimulated state, alpha has GDP bound and G protein inactive

• GPCR is active(performs GEF activity), alpha releases GDP to bind to GTP -> GTP causes a conformational change that releases G protein from receptor and dissociation of alpha G from beta and gamma pair G - they then can relay signals onwards

What happens to the alpha subunit when it becomes unstimulated?

Alpha then hydrolyzes the GTP to GDP and becomes inactive - bound to GDP

What do G-coupled receptors do?

Biological functions include smell and taste, light perception, neurotransmission, endocrine/exocrine glands, control of BP, exocytosis, cell growth and development, etc

Activation of heterotrimeric G-proteins act like…

GEF in GPCRs

What is cAMP?

cAMP acts like a second messenger in some systems and are made from ATP by adenylyl cyclase and destroyed by cyclic AMP phosphodiesterases

G-proteins and cAMP

• The alpha subunit in G proteins stimulates cAMP synthesis

• Gi proteins(inhibitory G protein) inhibit the cAMP synthesis

Cholera toxin

inhibits the switch-off mechanism of Gs and allows elevated cAMP [C] since the alpha unit is GTP bound and on

Pertussis toxin

inhibits the switch on of Gi that prevents the protein from interacting with others and keeps it in its inactive state

Remember that Gi is…

DIFFERENT FROM Gs!

Cellular responses mediated by cAMP

• Thyroid gland

• Adrenal cortex

• Ovary

• Muscle

• Bone

• Heart

• Liver

• Kidney

• Fat

cAMP dependant protein kinase

cAMP activates PKA that phosphorylates specific serines or threonines and regulates signal protein activity

• Inactive PKA has two catalytic subunits, cAMP binds and alters their shape and the two release from each other and go to target other proteins

• Regulatory proteins help localize the PKA to a specific complex via A-kinase anchoring proteins(AKAPs)

CREB(CRE-binding protein)

cAMP-dependent transcription factor

• Recognizes the cis-regulatory sequence called CRE found on many regulatory proteins activated by cAMP

• PKA activated, phosphorylates CREB which then recruits a transcriptional coactivator called CBP that stimulates the transcription of target genes

G proteins and phospholipid signalling

• activate the plasma-membrane-bound enzyme phospholipase-C(PLCβ) - it activates by Gq G protein that cleaves PI4,5P2 to make two products: IP3 and diacylglycerol

• IP3 goes through the ER and opens IP3-gated Ca channels, raising Ca ion[C] in the cytosol

• Diacylglycerol activates protein kinase C(PKC) is Ca-dependent and phosphorylates different proteins(similar to PKA)

Gq proteins activate PLCβ and calcium signalling

Phospholipase C(PLC) is activated by Gq -> inositol phospholipid signalling path

• PLC is activated by Gq which activates PI4,5P2 and makes two products

Protein Kinase C(PKC)

lacks Ca binding sites and needs to be altered to use Ca

• When Ca is increased initially by IP3, it alters PKC to face the plasma membrane where it is activated by Ca, DAG, and negative serine to target other proteins

Diacylglycerol(DAG)

It can be further cleaved to make arachidonic acid that can be used to synthesize prostaglandins or function as a signal molecule -> used in pain and inflammatory responses

Ca as a ubiquitous intracellular mediator

Many extracellular responses raise intracellular Ca[C] -> normally it is lower intracellular than extracellular

• Some processes outside the cell raise Ca[C] in the cell while some intracellular processes like IP3 receptors(GPCR mediated signals) increase Ca from inside the cell

Ryanodine receptors

in the ER membrane are activated by Ca and amplify Ca signals

Terminating the Ca2+ signal and maintaining low resting Ca

There are also Ca pumps in the ER and plasma membranes, which use ATP, that return calcium to intracellular stores

Calcium signalling -> spikes, puffs, and waves

IP3 and ryanodine are stimulated by low Ca[C] - Ca-induced calcium release is positive feedback -> a spark when IP3 is stimulated and Ca enters that then activates the next receptor and the next and so on, this sends a wave of Ca when the [C] of Ca is enough to activate nearby receptors and moves through the cytosol -> high [C] across the entire cell(rather than becoming more dilute) -> positive feedback

+/- feedback generates calcium waves

Eventually, the large [C] of Ca shuts down IP3 and ryanodine receptors that shut down Ca release and prevent more Ca from coming in -> Negative feedback -> Ca pumps remove the Ca

Ca oscillations of -/+ feedback

Eventually this (-) feedback will wear out and IP3 will trigger another wave of Ca -> these oscillations will continue to happen -> frequency reflects extracellular stimuli

Ca [C] in the cell and different systems

Different systems can use different amounts of Ca[C] to do different things - some need higher [C] to make one thing or lower [C] to make another -> depending on their sensitivity