Lecture 1:

• Cell communication

• Intracellular signaling pathway

• 4 components to cell communication

• Extracellular signalling

• Classes of intracellular signaling

  • contact-dependant

  • Paracrine

  • synaptic

  • endocrine

• Binding types:

  • cell surface

  • intracellular receptors

• Combination of biological responses

• cell types

• signal concentrations

• classes of cell surface receptors

  • ion-channel coupled

  • G-protein

  • enzyme coupled

• Second messengers

• Importance of intracellular signalling proteins

Lecture 2:

• Molecular Switches:

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

    • Many protein kinases are organized by kinase cascades that one gets activated, it activates the next, in a signal line -> adds more phosphate groups

      • Protein phosphatases can remove phosphate groups

    • Types of Kinases:

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

      • Tyr -> phosphorylates proteins on tyr

      • dual specificity -> 

  • 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

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

  • Regulation of monomeric GTPases: 

    • GAP GTPase activating protein -> causes the off state by hydrolyzing more GTP

    • GEF(guanine nucleotide exchange factor) -> causes the on state by eliminating GDP and allowing more GTP to bind

    • then GDI(GDP dissociation inhibitor)

  • Proteins can also be switched on/off by calcium of cAMP -> second messengers

  • Other post-translational modifications like methylation, acetylation, ubiquitylation

• Two negative inhibitory signals produce a positive effect

  • Most signals have activation and inhibition steps

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

• How to ensure signal response and specificity - lots of noise in a cell can cause signal molecules to bind/modify the wrong partner creating interference

  • High-affinity interactions that are highly specific -> docking sites - protein kinases that interact with specific amino acids

  • Signal threshold - reduction of background noise by the proteins by only responding to a set concentration of signals to activate it

  • Localization of specific signaling proteins to the same region of the cell

• Location: localizing the site for signalling proteins to ensure that a specific response is given while minimizing making unwanted responses with other pathways ex. Localizing in a place in the cell or within a larger protein

• Localizing by scaffold proteins - brings groups of interacting signalling proteins into signalling complexes often before the signal has been received  -> they are in the scaffold protein but are activated in a downstream manner when the signal protein activates the receptor

• Localizing at the site of activated receptors -> inactive receptor and proteins are not yet attached, then, when the signal molecule is attached and activates the receptor, it latches on all the other smaller complexes and sends a downstream signal

• Localizing with the help of phospholipids -> docking on the membrane and interacting with other signalling proteins when the receptor on the membrane is activated

• Importance of interaction domains:

  • Induced proximity -> signal triggers the assembly of complex proteins

  • Interaction domains -> areas of the proteins where they bind to other proteins

• Signal complex formation using modular interaction domains ex. Insulin receptors

• Different signalling pathways can vary in their properties

  • Response time(synaptic = fast, endocrine = slow), sensitivity(receptors, some need lower concentrations of a signal to activate, others higher[C]), dynamic range(adaptation mechanisms, some need specific signals other can have broader ones), persistence(+/- feedback), signal processing(ex. Gradual increase to abrupt), integration(many signals can activate one signal), coordination(one signal to a response that activates many signals)

• Signal integration and coincidence detectors

• Factors influencing response times

• Turnover (half-life) signalling molecules influence response time -> inhibition processes determine how quickly and great the response can be -> activation needs to be quickly revered to make rapid signalling possible

• Signal processing influences the properties of biological responses

  • 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

• Allosteric binding

• +/- feedback

• Importance of + feedback -> gradual increase in [C]

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

Lecture 3:

• Regulating signal sensitivity and dynamic range -> adaptation = the ability of a cell to respond to a wide range of signals [C]

  • Relative vs absolute [C]

• GPCR = G-protein coupled receptor

  • Largest family of cell surface receptors -> one ligand can bind to many GPCRs and one GCPR to many ligand types - all are 7 transmembrane(folded in the membrane 7 times) and signals through heterotrimeric G proteins

    • Rhodopsin is a member of the G protein family

• GCPR ligand diversity

  • Nucleotide - A, cAMP, melatonin

  • Biogenic amines - dopamine, adrenaline, ACTH, serotonin, histamine

  • Lipid-based

  • Excitatory amino acids and ions

  • Retinal based - vit. A

• Heterotrimeric G-proteins: all have 3 subunits of alpha, beta, and gamma

  • Couples the receptor to enzymes or ion channels in the membrane 

  • 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

  • Alpha then hydrolyzes the GTP to GDP and becomes inactive

• G-coupled receptors

  • 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

• G-proteins and cAMP -> cAMP act like a second messenger in some systems and are made from ATP by adenylyl cyclase and destroyed by cyclic AMP phosphodiesterases

  • 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

  • Gi is different from Gs!

• Cellular responses mediated by cAMP

  • Thyroid gland -> TSH secretion and synthesis

  • Adrenal cortex -> ACTH and cortisol secretion

  • Ovary -> LH and progesterone secretion

  • Muscle -> Adrenaline and glycogen breakdown

  • Boen -> parathormone and bone reabsorption

  • Heart -> adrenaline and increase HR + contractions

  • Liver -> glucagon and glycogen breakdown

  • Kidney -> vasopressin and water reabsorption

  • Fat -> adrenaline, ACTH, glucagon, TSH and triglyceride breakdown

• 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

  • Some cell responses that use GPCRs that activate PLC are liver(vasopressin), pancreas(acetylcholine), smooth muscle(acetylcholine), and blood patelets(thrombin)

  • Many GCPRs affect G proteins that 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 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 and oscillations

  • 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

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

    • 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