BMS100 ClinPhys Intracellular Signaling.docx

BMS100: CLINCAL PHYSIOLOGY REVIEW NOTES INTRACELLULAR SIGNALING AND THE CELL MEMBRANE Transduction: intracellular events that transform the extracellular signal into an intracellular signal Receptors in cell membrane detect extracellular signal Also in the membrane are source of, accumulation and r...

BMS100: CLINCAL PHYSIOLOGY REVIEW NOTES INTRACELLULAR SIGNALING AND THE CELL MEMBRANE Transduction: intracellular events that transform the extracellular signal into an intracellular signal Receptors in cell membrane detect extracellular signal Also in the membrane are source of, accumulation and regulatory sites for 2nd messengers Amplification: single activated receptor that results in many molecules of 2nd messenger Signal termination can be due to: Inactivation of a receptor (no 1st messenger) or receptor-associated effectors Degradation or removal of 2nd messenger Negative feedback Model of intracellular signaling Activation Steps: Concentration of 1st messenger increases (ligand) Binds to cell membrane receptor Activated cell membrane receptor results in activation of an intracellular protein associated with the receptor That protein activated a mechanism (ex. enzyme) that increased the intracellular concentration of the active form of a second messenger Second messenger binds to and activates another protein which will activate or inactive other signalling cascades (effectors) Effectors examples: protein kinases, transcription factors Inactivation Steps: Concentration of 1st messenger decreases (ligand) Cell membrane receptor no longer activated; protein associated with receptor becomes inactive (often inactivates itself) Protein/enzyme no longer produces or increases concentration of the second messenger and another mechanism decreases/inactivates second messenger Protein and effectors are no longer activated Types of Cell Membrane Receptors – 3 classes ION-CHANNEL-COUPLED RECEPTORS Receptors can open ion-channels after they bind a ligand (1st messenger); when channel opens it can become more: Inside-positive – if channel allows sodium to enter = depolarization Inside-negative – if channel allows more potassium to leave = hyperpolarization Binding to calmodulin – if channel allows calcium to enter cytosol Na+/K+-ATPase and K+ channels cell membrane is inside-negative with low cytosolic concentration of sodium and high concentration of K+; sodium wants to diffuse into cell if channel opens G-PROTEIN COUPLED RECEPTORS Largest family: almost half medications act on these receptors or pathways How it works: receptor activation leads to activation of a protein that binds to a guanine nucleotide activated G-protein will modify the activity of an enzyme G-protein is on a timer – has intrinsic GTP-ase activity; when GTP hydrolyzed to GDP then the G-protein is inactive Structure: Receptor type: integral transmembrane protein (spans membrane 7 times) 3 protein subunits – alpha (α), beta (β), gamma (γ) Unstimulated (inactive) – α bound to GDP; and βγ is bound to α Stimulated (active) – α subunit releases GDP replacing it with GTP and α subunit disengages from the βγ subunits When the α unit hydrolyzes GTPGDP it becomes inactivated again; βγ subunit can also sometimes activate signals Gs GPCR How it works: release of PK A inhibitors allowing calcium to move down its concentration gradient from storage to cytosol cAMP – inactivation of cAMP results when it is converted to 5’-AMP by cyclic AMP phosphodiesterase Steps: Ligand binds to receptor associated with Gs G-protein Gs releases GDP and binds GTP at α subunit – βγ subunit detaches from G-protein Gs binds and activates adenylyl cyclase – membrane-bound enzyme that converts ATP to cAMP cAMP binds to protein kinase A (PK A) – binds to inhibitors of PK A which detach, and allow active parts of PK A to work PK A phosphorylates a multitude of effector proteins Gq GPCR How it works: uses Ca2+, IP3 and DAG as 2nd messenger systems IP3 – causes release of Ca2+ from where it is stored in ER Ca2+ - can bind and activate proteins to modulate a number of effectors Ca2+-Calmodulin interactions Resting cell has 0.1mmol of Ca2+ in cytosol and 1-3 mmol in ER and in extracellular space = concentration gradient is high and wants to enter cytosol Activated cell can reach Ca2+ concentrations of 10mmol or more When Ca2+ in cytosol increases = bind to calcium-binding proteins in cytosol Each calmodulin binds to 4 Ca2+ before it is activated = bind to effectors such as calmodulin kinases Steps: Ligand binds to receptor associated with Gq G-protein Gq-alpha activates phospholipase C Phospholipase C cleaves membrane lipid (PIP2) into IP3 and DAG – IP3 is water soluble and enters cytosol; DAG is lipid soluble and stays in cell membrane IP3 activates a Ca2+-release channel in ER movement of Ca2+ from ER into cytosol Ca2+ and DAG work to activate membrane-bound PK C PK C (2nd-messenger-activated effector) can modulate other effectors and Ca2+ can bind to other molecules like calmodulin Gi GPCR Ubiquitous inhibitory G-protein that downregulates the activity of Gs Gi-α => inactivates adenylyl cyclase Gi-βγ => opens a K+ channel Opening of K+ channels brings the cell closer to its Nernst potential for K+ -90 mV is the Nernst potential for K+ - very negative membrane potential which tends to cause most cells to be “less” activated ENZYME-COUPLED RECEPTORS Enzyme-couple receptors: transmembrane proteins with ligand-binding domain on outer surface of plasma membrane (usually 1 transmembrane domain) Cytosolic domain has either: Intrinsic enzyme activity – most common receptor tyrosine kinase Direct association with enzyme Receptor tyrosine kinases Has intrinsic kinase activity – phosphorylates itself on specific residues of intracellular face of receptor = further signaling Binding of ligand dimerizes the receptor and activates a tyrosine kinase within receptor Ligand examples: insulin, growth factors, cytokines Steps for RTK enzyme-coupled receptors: Ligand binds to receptor monomers Receptor dimerizes and each half phosphorylates the tyrosine residue on other half Signaling proteins bind to the phosphorylated receptor and can become activated = signal cascade Signaling options for RTKs: Phospholipase C Ras cascade *most common* Ras (intracellular G-protein) encounters activated RTK and binds to GTP = activation Ras activates Raf (small plasma-membrane-associated G-protein) Activated Raf activation of MAP kinases Ras inactivates itself by cleaving GTP to GDP PI-3-Kinase (PI3K) Akt System – key to insulin signaling RTK activated causes activation of nearby phosphoinositide-3-kinase (PI3K) (Ras can also directly activate) PI3K attaches additional phosphate to PIP2 (membrane lipid) to form PIP3 (2nd messenger) PIP3 accumulates and forms lipid rafts in membrane Akt and PDK1 (kinases from cytosol) accumulate and cluster together at the site of the PIP3 rafts; PDK1 becomes activated by PIP3 Activated PDK1 activates Akt by phosphorylation Akt is the effector influencing intracellular targets Nitric Oxide Signaling Nitric oxide (NO) key mediator that relaxes smooth muscle in blood vessels and visceral organs Small, hydrophobic gas that diffuses easily and quickly through cell membranes = can mediate signals within cell produced or diffuse into another cell Produced by action of NO synthase on L-arginine Increases in cytosolic calcium can activate Nos NO degraded rapidly since it reacts with oxygen and water It is a second messenger; one of the only that can diffuse across cell membrane but only local effects since it degrades quickly Nitric Oxide-Mediated signaling steps: Cytosolic calcium increases Increased intracellular calcium activates NOs NOs produce NO from L-arginine NO binds and activates guanylyl cyclase (GC) production of cGMP (second messenger) from GTP Elevations of cytosolic cGMP activates a protein kinase (PKG) changes cellular activity Many cases results in disengagement of myosin from actin in smooth muscle = smooth muscle relaxation