E2 - PM Receptors & Cell Signaling

Discuss the role of G proteins and cAMP in signal transduction. 1) What are they & their function? 2) What are the 3 main ways this process is controlled/regulated? 3) Give the 6 steps of this signal transduction process (starting with inactive state).

1) A G-protein coupled receptor (GTPase) that uses cAMP as secondary messenger; For stimulatory/inhibitory hormone that can't penetrate PM & needs exterior binding site. (G protein = α, β, & γ subunits, which is then linked to a receptor = G protein-linked (coupled) receptor) 2) a. Control signaling system by using a phosphodiesterase enzyme which hydrolizes cAMP into AMP. Keeps signal from always being on. b. G-protein has finite half-life. c. G-protein-linked receptor can bind to inhibitory receptor and stay inactive as long as hormone receptor is activated. 3) 6 Steps: 1. Inactive state: α, β & γ subunits are present as complex, with GDP bound to the α subunit. 2. (3) When ligand binds receptor, it binds and activates G protein. Activation causes displacement of GDP by GTP and dissociation of α-subunit. (G proteins respond quickly to changes in ligand concentration because they remain active for only a short time before α-subunit hydrolyzes its bound GTP & converts to inactive state.) 3. (2) α-subunit then binds to/activates adenylyl cyclase protein, which synthesizes cAMP: ATP → (AC) → PP(i) + cAMP (cAMP important for many events, a few of & appears to have 1 main intracellular target, enzyme protein kinase A (PKA phosphorylates wide variety of proteins).) 4. (3) Activation ends when ligand leaves receptor; GTP is hydrolyzed to GDP (by GTPase activity of α-subunit; aka + H2O) and α-subunit dissociates from adenylyl cyclase. 5. Adenylyl cyclase then reverts to inactive form, the α-subunit re-attaches with βγ-complex. 6. cAMP molecules in cytosol are hydrolyzed to AMP by phosphodiesterase. (Once G protein is inactive, adenylyl cyclase ceases to make cAMP. cAMP levels would still remain elevated in cell if not for phosphodiesterase, which degrades cAMP to further ensures that signal transduction pathway will shut down promptly when concentration of ligand outside the cell declines.)

Discuss the role of G proteins, DAG and IP3 in signal transduction: 1) What are they & their function? 2) Give the 4 basic steps in this process.

1) A G-protein coupled receptor that uses DAG & IP₃ secondary messengers. For stimulatory hormone that can't penetrate PM, needs exterior binding site. - DAG: a. DAG stimulates protein kinase C. b. Protein kinase C phosphorylates another kinase, etc. = cascade. - IP3: (finite half-life) a. IP3 binds to Ca channel on ER, stimulating flood of Ca into cell. b. Flood of Ca will eventually have another cellular response via the Ca/calmodulin complex. (Lipid derived secondary messengers) 2) Steps: 1. Ligand binds to receptor, activating it. G protein attaches to complex, causing displacement of GDP by GTP and of α subunit. 2. α subunit (with GTP) binds to phospholipase C, activating it and causing cleavage of PIP2 → IP3 + DAG. 3. IP3 released into cytosol, triggering calcium release. 4. DAG remains in membrane where it activates protein kinase C.

Discuss the synthesis and function of cGMP coupled signal transduction. 1) What is it? (give function) 2) In what area(s) can the cGMP process commonly be seen?

1) A G-protein coupled receptor that uses cGMP as secondary messenger. For stimulatory hormone that can't penetrate PM, needs exterior binding site. - Basic Info: a. Guanylate cylcase (GC) usually stimulated by NO. b. GC catalyzes cGMP synthesis, converting new GTP → cGMP (cGMP produced continuously) c. cGMP activates protein kinase G (PKG), enabling it to phosphorylate substrates. d. Unlike PKA, PKG is activated, but catalytic and regulatory subunits don't dissociate. e. Phosphodiesterase degrades cGMP 2) A common regulator of ion channel conductance (i.e. calcium movement) glycogenolysis, and cellular apoptosis. Transduction Example: (eye) 1. Rhodopsin acts as receptor and stimulated by photon. 2. Undergoes conformational change and activates G Protein (transducin). 3. Transducin stimulates cGMP phosphodiesterase. 4. Phosphodiesterase converts cGMP to GMP. 5. GMP opens up ion channels and causes rods to depolarize. (Rods and cones are specialized neurons) *It also relaxes smooth muscle tissues. In blood vessels, relaxation of vascular smooth muscle leads to vasodilation and increased blood flow.

Discuss the role of tyrosine kinases in: 1) Signal transduction 2) What is the basic process these receptors go through to be/when activated? (Give the nature of MAP kinase cascade)

1) A Protein-Tyrosine Kinases Receptor (not g-protein coupled): a. Receptor Tyrosine Kinases (RTKs) not only function as receptors, but are also protein kinases themselves. b. Exists as 2 free-floating subunits of 2 smaller subunits in absence of ligands. c. Target for phosphorylation is tyrosine AA in cytoplasm. d. Many RTKs trigger chain of events inside cell that ultimately lead to cell growth, proliferation, or specialization of cells. - RTK Ex: Insulin receptor, nerve growth factor receptor, & epidermal growth factor (EGF) receptor. 2) Process: a. When ligand binds, 2 subunits dimerize and autophosphorylate themselves or phosphorylate a target that interacts with them. b. Ras, a G protein they phosphorylate, is a single polypeptide that's similar to an α subunit in terms of structure. c. When phosphorylated (GDP→GTP), Ras is activated and then goes on to activate Raf kinase. d. Raf then activates MAP kinase cascade through MEK. c. MAP cascade will eventually stimulate some cell function (i.e. activation of transcription & translation) *Ras mutations inhibit its ability to hydrolyze GTP and Ras is trapped in "ON" position. Everything downstream from Ras will be continuously on and can result in cancer. Ras is a major oncogene.

Give the 2 groups of cell surface receptors and their sub-groups.

1) G-Protein-Coupled Receptors (GPCRs): a. Using cAMP secondary messengers b. Using cGMP secondary messengers c. Using DAG & IP₃ secondary messengers 2) Receptor Protein-Tyrosine Kinases (RPK)

Discuss the effect of cholera and pertussis toxins on G proteins.

Cholera: Inhibits Gα GTPase activity of intestinal epithelium. Causes cells to secrete large volumes of fluid into intestinal lumen (what kicks off life threatening diarrhea). Pertussis (Whooping Cough): Inactivates Gα GTPase, leading to lack of host defensive response to the bacteria (compromises immune system allowing bacteria to grow).

1) Discuss convergence, divergence, and crosstalk among signaling pathways. 2) Give examples of each.

Convergence: Different signals from different receptors, but end up in same spot. Ex: Convergence of different signals as Ras, which then goes on to activate MAP kinase cascade. Divergence: Uses a messenger and once generated, can activate many different processes of cell in different locations. Ex: cAMP as messenger which activates kinases, etc. Crosstalk: One or more component of signal transduction pathway affect a different pathway. There are often shared components that interact w/ more than one.

G-Proteins: 1) Describe Use/Function 2) Describe Structure

G-Proteins = GTPase 1) Use/Function: a. Family of proteins involved in transmitting chemical signals from outside to inside of cell. b. Function as molecular switches. c. Activity regulated by factors that control their ability to bind to & hydrolyze GTP → GDP. d. "ON" when G-Protein binds to GTP; "OFF" when G-Protein hydrolyzes GTP + H2O → GDP + Pi. *GTP has finite half-life since will be hydrolyzed 2) Structure: a. Lipid-linked to bilayer on cytoplasmic side. b. Composed of 3 subunits: (1) α-unit - binds/hydrolyzes GTP/GDP (2) β-unit - the inhibitor (3) γ-unit

List at least two types of receptor tyrosine kinases.

Over 50 RTKs found, including: 1. Insulin receptor 2. Epidermal growth factor 3. Interferon receptor 4. Interleukin receptor

Discuss the role, synthesis and mechanism of nitric oxide (NO). - Specifically in muscle activity

Role: a. Toxic, short-lived gas molecule. b. Important signaling molecule in cardiovascular system. Synthesis: a. Produced by use of enzyme NO synthase by 1 of 2 ways: (1) L-arginine + O2 + NADPH → NO (2) Nitroglycerine → NO Mechanism (endothelial cells): Endothelial Cell → Smooth Muscle Cell 1) ACh binds to G protein-linked receptors, activate phosphoinositide signaling pathway, causing IP3 + DAG created by endothelial cells. 2) IP3 causes release of Ca from ER 3) Ca binds to calmodulin & this complex stimulates NO synthase to produce NO. 4) NO gas diffuses through PM, passing through PM of endothelial cell to adjacent smooth muscle cells. 5) Inside smooth muscle cell, NO activates guanylyl cyclase, catalyzing formation of cGMP 6) Increase in [cGMP] activates PKG, inducing relaxation in muscle by catalyzing phosphorylation of muscle proteins.