Cell Signalling

• • The two major receptor types are: cell-surface receptors, which bind to hydrophilic signalling ligands because these ligands cannot cross the plasma membrane; and intramembrane receptors, which bind to hydrophobic ligands because these ligands can be transported by polar carrier proteins in aqueous space and diffuse through plasma membrane to bind to these receptors in cytosol or nucleus.
• • The four major modes of cell signalling are: contact-dependent signalling, where membrane-bound signal of one cell directly makes contact with intermembrane receptor of neighbouring cell; paracrine (autocrine) signalling, where cells secrete local mediator molecules in extracellular space that bind to receptors on target cells; synaptic signalling, long distance signalling from neuron to target cell by releasing neurotransmitters at synapse; endocrine signalling, long distance signalling involving secreted hormones travelling to receptors on target cells around body via circulatory system.
• • Cells are programmed to respond to specific combinations of extracellular signals by different signals enabling the cell responses for specific activities, such as combination of signals for cell survival and growth. Because without combination of extracellular signals, cells receiving no signals result to state of apoptosis.
• • The three major classes of cell-surface receptors are: ion-channel receptor, which enables signalling between neurons & other electrically-excitable cells through letting ions through; G-protein coupled receptors, which receive signal, activates G-protein that activates receptors & itself to further activate associated enzymes; enzyme-coupled receptors, which receive signals to turn on receptor's dimerisation by intrinsic enzymatic activity or associated enzymes on receptor.
• • Cell-surface receptors relay signals from intracellular signalling molecules by molecular switches of signalling by phosphorylation and signalling by GTP-binding (i.e. monomeric GTPase).
  • This is because phosphorylation (kinase) & dephosphorylation (phosphotase) can both activate/inactivate signal receptors for signal out or no signal out of cell. While GEF exchanging GDP for GTP allows GTP to bind & activate signal receptors for signal out, and GAP hydrolysing GTP allows GDP to bind & inactivate signal receptors for no signal out.
• • Intracellular signalling complexes form at activated cell-surface receptors by upstream signal activating protein kinase (cell-surface receptor), which phosphorylates inhibitor proteins to inhibit inhibitor activity. Thereby, activating the transcription regulators (signalling complexes) previously inhibited by inhibitor proteins.
• • The relationship between a signal & a cellular response vary in different contexts by: cellular response to extracellular signal being slow, if involving changes in gene expression or protein synthesis (e.g. cell growth & division) or rapid, if only changes in effector protein (e.g. changes for cell movement); and cellular response to signal being positive feedback, if downstream protein amplifies/increases activity of upstream protein activated by signal, or negative feedback, if downstream protein inhibits/decreases activity of upstream protein activated by signal.
• • The structure of G-protein coupled receptors involve: 7-pass transmembrane proteins; small extracellular domain, for binding small ligands (e.g. neurotransmitters); large extracellular domain, for binding large ligands (e.g. proteins); ligand binding site, deep within protein & consists of amino acids from transmembrane segments.
• • The structure of G-protein is a trimeric GTP-binding protein, consisting of: alpha & gamma subunits, that are membrane bound; and beta subunit, that is cytosolic.
  • G-protein is activated when its alpha subunit binds to GTP, and inactivated when its alpha subunit binds to GDP.
• • The activation of G-protein & GPCR occur by a signal binding to GPCR at cell surface, triggering a conformational change in GPCR. So receptor can bind to & alter conformation in G proteins, causing AH domain of alpha subunit to move outward & open a nucleotide binding site. This promotes dissociation of GDP from alpha subunit, so it binds to GTP to close nucleotide binding site. This activated alpha subunit triggers alpha-subunit conformational change, so it dissociates from GPCR & activated beta-gamma subunit. Activated subunits each regulate downstream signal activity.
• • The activation of G-protein & GPCR can be continuous because as long as signal is still bound to GPCR, the receptor stays active. Hence, it can catalyse the activation of many G-proteins per signal.
• • An activated G-protein can regulate gene expression by activating adenyl cyclase (AC) that converts ATP into cAMP. This is because cAMP then binds to PKA at regulatory subunits, to change its conformation & release catalytic PKA subunits. Catalytic subunits are then transported into nucleus via nuclear pore, to phosphorylate/activate CREB proteins. Activated CREB recruits CREB binding protein (CBP) & this whole complex binds onto cAMP response element on DNA, for target gene expression.
• • An activated G-protein can regulate ion flux across a membrane by acetylcholine (Ach) binding to GPCR to decrease heart firing. This is because the activated G-protein alpha subunit would then inhibit AC, so less cAMP binds to PKA, while activated G-protein beta-gamma subunit activates K+ ion channels.
• • Light activates GPCR signalling in photoreceptors by light activating rhodopsin photoreceptor that activates G-protein for cell hyperpolarisation for vision. This is because light causes rhodopsin conformational change which activates G-alpha subunits (via GTP), that then activates cGMP phosphodiesterase. This enzyme hydrolyses cGMP resulting in less cGMP concentration, closing cGMP-gated Na+ channels for cell hyperpolarisation. This allows low neurotransmitter (NT) release rate, so ON bipolar cells activated due to no inhibition from NT. ON cell transmits signal to brain for detecting light.
• • When there is no light to activate GPCR in rhodopsin, cell depolarisation occurs by activated glutamate (NT) exciting OFF bipolar cells. This is because in darkness, rhodopsin kinase phosphorylates rhodopsin causing arrestin to bind & inhibit P-rhodopsin. RGS protein is then released to bind to G-protein, which hydrolyses GTP to GDP for inactivation. This causes high cGMP concentration, so cGMP-gated Na+ channels are open for cell depolarisation. This allows glutamte NT to be transmitted at a high rate to inhibit ON bipolar cells & activate OFF bipolar cells for no signal transmitted to brain.
• • The structural characteristics of receptor tyrosine kinase (RTK's) are: variable extracellular domain, domains for ligand binding; single transmembrane domain, embedded in plasma membrane; variable intracellular domain, where tyrosine kinase domains are located.
• • The mechanism of action of RTK is the trans-autophosphorylation mechanism between two tyrosine kinase domains of RTK's. This occurs by dimer ligand binding to two RTK's, or by ligand inducing RTK conformational change for two RTK's, for dimerisation that allows autophosphorylation of the two RTK's by tyrosine kinase domain. This generates binding/docking sites, of phosphorylated-Tyr residue, for other signalling molecules to activate signal relay for downstream signals.
• • The Ras superfamily is a superfamily of GTPases. The families in superfamily are: Ras family; Rho family; Arf family; Rab family; and Ran family.
• • The Ras superfamily of monomeric GTPases are activated by RTK being activated by trans-autophosphorylation through binding to extracellular signal. Adaptor protein, Grb2, then binds to a P-Tyr residue & another specific amino acid on activated RTK by its SH2 domain. The SH3 domain of activated Grb2 then binds to Sos protein, which activates Ras-GEF domain of Sos. Ras-GEF domain exchanges Ras-GDP bound to Ras for Ras-GTP which activates Ras. Activated Ras then activates other downstream signals.
• • Activation of Ras leads to the activation of mitogen activated protein kinase (MAPK) pathway by Ras changing the conformation of Raf to activate it. Activated Raf then phosphorylates Mek for activation. Activated Mek then phosphorylates Erk for activation. Activated Erk then phosphorylates proteins or transcription regulators for changes in protein activity & gene expression.
• • Activation of Ras leads to the activation of MAPK pathway because the MAPK pathway sustains the short-period RTK-Ras activation to a longer period. Since phosphotase in cells rapidly remove phosphate from Tyr in RTK, while Ras-GAP's rapidly hydrolyse Ras-GTP to Ras-GDP to inactivate Ras.
• • The common responses by Ras protein activating MAPK pathway are cell proliferation and cell differentiation.
• • The ways to study RTK signalling include increasing expression of dominant-negative receptors with truncated intracellular domain. This ensures that no signal transduction can be induced by ligand binding to RTK, so that effects of inhibiting RTK signalling in animal cells can be examined.
• • The role of overlapping signalling pathways is to modulate and coordinate signalling responses for overall cell function.
• • Some enzyme-coupled receptors associate with cytoplasmic tyrosine kinases by the binding of its SH2 domain to phosphorylated Tyr residue on RTK. This is because SH2 domains have a site for P-Tyr residue as well as a separate site for a specific amino acid residue, allowing high specificity in binding to RTK. These enzymes/proteins associate with RTK for activation to act as intracellular relays of signal received by extracellular domain of RTK.
• • The signal proteins of TGFB superfamily activates pathways by TGFB signal proteins binding to type I & type II TGFB receptor homodimers with S/T kinase domain, usually on the plasma membrane. This binding forms endosomes of clathrin-coated vesicles, so Smad signalling starts from endosomal membrane. Type II homodimer constitutively active & phosphorylates type I homodimer, activating the whole TGFB receptor complex to then phosphorylate R-Smads. Two activated R-Smad recruits a co-Smad, to form trimeric Smad complex.
• • Trimeric Smad complex being translocated into nucleus forms transcription regulatory complex with other complexes. This transcription regulatory complex then binds to cis-regulatory sequences to initiate target gene transcription.
• • The common responses from signalling of TGFB include: inhibition of proliferation; cell specialisation; differentiation; extracellular matrix production; tissue repair; EMT/fibrosis; and cell death.
• • An example of regulated proteolysis that controls the activity & location of a latent transcription factor is the Wnt signalling pathway (or canonical pathway), which controls activity & location of beta-catenin.
• • With no signals in the Wnt signalling pathway, it controls the activity and location of beta catenin by causing degradation of beta catenin for no transcription of Wnt target genes. It does this by allowing beta-catenin destruction complex (APC, axin, active GSK3 & active CK1) to form around beta-catenin. The active GSK3 & CK1 phosphorylates beta-catenin for ubiquitylation by E3-ubiquitin ligase, which targets it for degradation in proteasome. This prevents the activation of Wnt target genes, because Groucho repressor still binds to LEF1/TCF transcription factor.
• • With Wnt signal binding to Frizzled receptor for Wnt gene transcription, it controls the activity & location of beta-catenin by allowing beta-catenin to bind to LEF1/TCF as co-activator. This occurs by Wnt signal binding to Frizzled & LRP receptor at the same time for activation. So active GSK3 & CK1 then phosphorylates LRP tail & Frizzled activates Dishevelled, these bind to axin to inactivate & degrade it, taking apart beta-catenin destruction complex. Stable beta-catenin can accumulate & translocate into the nucleus, where it binds to LEF1/TCF & displaces Groucho.