L7 GPCR

Objectives

Discuss the basic structure and general signaling of GPCRs

Describe the role and types of G proteins

Discuss the signaling mechanism for GPCRs that couple to Gαs and Gαi and role of PKA

Discuss the signaling mechanism for GPCRs that couple to Gαq, Gα12/13 proteins

Describe the role of Ras G proteins

  1. What are Metabotropic receptors and give an example.

    • membrane receptors that trigger metabolic processes to regulate cell activity

    • G Protein Coupled Receptors (GPCR)

  2. What is GPCR’s nickname and why?

    • Serpentine Receptors

    • 7 Transmembrane-Spanning Receptors (7TM)

  3. Why are GPCRs the largest family of receptors expressed in human genome

    • > 800

  4. Why are GPCRs the most prominent therapeutic target family

    • Beta-adrenergic receptor agonists & antagonists

    • Angiotensin receptor blockers

    • Histamine H1 receptor antagonists (antihistamines)

    • Dopamine & Serotonin receptor agonists

    • Opioid receptor antagonists

  5. Why did Robert J. Lefkowitz and Brian K. Kobilka win the Nobel Prize in Chemistry 2012?

    • Studies of “G-Protein-Coupled Receptors"

  6. What are the 3 main classes based on Sequence Homology

    • A, B, and C

  7. What are the 6 classes based on Structure and Function

    • GRAFS: Glutamate, Rhodopsin, Adhesion, Frizzled, Secretin

  8. What are the domains of GPCRs

    • Extracellular (EC) — Ligand Binding

    • (7) Trans-Membrane Helices (TM)

    • Intracellular (IC) — signaling

  9. What does the intracellular domain interact with?

    • G-proteins

  10. What are the “First Messengers” for GPCR signaling?

    • Hormones and Neurotransmitters

  11. What are the “Second Messengers” for GPCR signaling?

    • cAMP

    • cGMP

    • IP3 — Inositol Triphosphate

    • DAG — Diacyl Glycerol

    • Ca++

  12. What is the role of the G Protein?

    • a membrane-associated protein that regulates an enzyme which produces a second messenger and makes a signal relay

  13. What happens after a signal relay?

    • activation of Ser/Thr Kinases → Phosphorylation of substrates → amplification of biological response of cells

  14. Explain the general signal transduction mechanism

    • Agonist → GPCR → Effector (target enzymes) → Protein kinases → Cellular Effect

  15. What are examples of target enzymes?

    • Guanylyl Cyclase, Adenylyl Cyclase, Phospholipase C

  16. What energy occurs after the first messenger binds to the receptor?

    • GDP → GTP

  17. What binds to GTP?

    • G Proteins

  18. What type of activity does G Proteins undergo

    • GTPase activity

  19. What 2 states does G proteins cycle between?

    • GDP bound — inactive

    • GTP bound — active

  20. What are the 2 families of G proteins

    • Heteromeric G Proteins

    • Ras Superfamily G proteins

      • “small G proteins / GTPases”

  21. What are the 3 subunits of Heterotrimeric G Proteins

    • Alpha, Beta, Gamma

      • all join together and bind to GDP when inactive

  22. What type of receptors do Heterotrimeric G proteins use

    • G-protein linked receptors

  23. What is the purpose of Heterotrimeric G proteins

    • regulates second messengers

  24. Describe the monomers of Ras superfamily G proteins

    • resemble Alpha subunit of Heterotrimeric G proteins

  25. What type of receptors does “Small GTPases” use

    • Catalytic receptors

  26. What are the 4 categories of Alpha subunits in Heterotrimeric G Proteins and their main effectors

    • G-alphas — stimulate Adenylyl Cyclase → increase cAMP formation

    • G-alphai — inhibit Adenylyl Cyclase → decrease cAMP formation

    • G-alphaq — activate Phospholipase C (PLC) → increase production of Inositol Trisphosphate (IP3) and Diacylglycerol (DAG)

    • G-alpha12/13 — activate PLC-Epsilon, Phospholipase D, Small GTPase, RhoGEF-Rho

  27. Why did Earl Sutherland win the Novel Prize in Physiology or Medicine 1971

    • for his discoveries concerning the mechanisms of the action of hormones

  28. Explain the G-alphas / Gs - cAMP signaling pathway

    1. Unoccupied receptor does not interact with Gs protein

    2. Occupied receptor changes shape and interacts with Gs protein which releases GDP → binds to GTP

    3. Alpha subunit of Gs protein dissociates and activates Adenylyl Cyclase

    4. When hormone is no longer present, receptor rests while the GTP on the Alpha subunit Hydrolyzes to GDP and Adenylyl Cyclase is deactivated

  29. What targets G-Alphas?

    • Cholera Toxin

  30. What is Cholera Toxin produced by?

    • Vibrio Cholera to infect intestinal epithelial cells

  31. What happens when Cholera Toxin targets G-Alphas?

    1. G-Alphasb unit is modified → inhibits GTPase activity → GTP cannot be hydrolyzed

    2. Adenylyl Cyclase remains active indefinitely

    3. excess cAMP → excessive water and Cl- in gut

    4. severe diarrhea and dehydration

  32. What protein kinase is dependent/target of cAMP?

    • Protein Kinase A (PKA)

  33. Explain the structure of an inactive PKA

    • regulatory and inactive catalytic subunits

  34. Explain what happens when PKA and cAMP binds

    • cAMP binds to the regulatory subunits of PKA → releases active Catalytic subunits → PKA mediated protein phosphorylation

  35. Explain PKA mediated Protein Phosphorylation

    1. PKA transfers a Phosphate from ATP to Serine, Threonine, or Tyrosine of another protein

  36. How does PKA mediate FAST Signaling Responses

    • phosphorylates intracellular enzymes or ion channels

  37. How does PKA mediate SLOW Signaling Responses

    • regulating gene transcription

  38. How does active PKA translocate from cytoplasm → nucleus?

    • phosphorylates transcription factor cAMP response element binding protein (CREB)

  39. What targets G-Alphai?

    • Pertussis Toxin

  40. What is Pertussis Toxin produced by?

    • Bordetella pertussis which infects the respiratory tract and causes pertussis or whooping cough

  41. What happens when Pertussis Toxin targets G-Alphai?

    1. G-Alphai is inhibited

    2. Adenylyl Cyclase remains active indefinitely

    3. Excess cAMP

    4. coughing, vomiting, dehydration

  42. Adenylyl cyclase is activated by a G protein. Which of the following second messengers will be generated ?

    • cAMP

  43. Explain the G-Alphaq (Gq) subunit IP3 signaling pathway

    1. Hormone binds to specific receptor

    2. Occupied receptor interacts with Gq protein

    3. Gq’s Alpha subunit releases GDP to bind to GTP

    4. Alpha subunit dissociates from Beta and Gamma to activate Phospholipase C

    5. Active Phospholipase C cleaves PIP2 → IP3 and DAG

    6. IP3 binds to ER → releases sequestered Ca++

    7. Protein Kinase C catalyzes phosphorylation of cellular proteins that mediate cell responses

  44. What is Calmodulin (CaM)

    • calcium binding protein that is essential for Ca++ dependent enzymes

  45. Explain the actions of CaM

    1. Ca++ is released from ER in response to neurotransmitters/hormones binding to cell surface receptors

    2. CaM-Ca++ complex forms

    3. activates Ca++ dependent enzymes

  46. What second messengers are produced from membrane phosphatidylinositol 4,5-bisphosphate by phospholipase C?

    • IP3 and DAG

  47. Which of the following is activated by diacylglycerol (DAG) and Ca2+ and produces protein phosphorylation?

    • Protein Kinase C (PKC)

  48. What is activated when G-Alpha12/13is signaling

    • Phospholipase C-Epsilon (similar to G-Alphaq)

    • Phospholipase D

    • Members of Ras family of small GTPases

  49. What physiological roles does G-Alpha12/13 has?

    • Cell growth and apoptosis

  50. Where can disrupted/abnormal G-Alpha12/13 signaling occur?

    • Leukemia cells and Malignant Cell Transformation & Metastasis

  51. What is activated when G-BetaGamma subunit is signaling

    1. K+ channels

    2. activation/inhibition of Adenylyl Cyclase

    3. Phospholipase C (PLC)

    4. GPCR Kinases (GRKs)

  52. GPCR ligands that activate G alphai (Gαi or Gi) proteins

    • inhibit adenylyl cyclase and decrease cAMP

  53. What proteins are homologous to the Alpha subunits of Heterotrimeric G proteins

    • Ras G Proteins

  54. What happens when Ras G proteins are activated by GTP

    • stimulate the MAP kinase cascade of serine/threonine phosphorylations → activation of gene transcription

  55. What does Ras G proteins stimulate?

    • Proliferation

  56. What happens then there’s a dysfunction with Ras G proteins?

    • upregulated cell division and malignancy

  57. Explain the steps of Ras Signaling

    1. ligand binding to catalytic R's can cause phosphorylation of tyrosine residues within the R

    2. specific SH2-containing adapter proteins dock at the phospho-tyrosine residues; Ras-specific guanine exchange factor (GEF) SOS joins

    3. Ras binds to SH2-containing proteins docked to phospho-tyrosines; GTP replaces GDP, activating Ras

    4. Ras-GTP promotes binding and phosphorylation of RAF (serine protein kinase; aka MAPKKK)

    5. a phosphorylation cascade then includes MAPKK (MEK), which, when activated, phosphorylates MAPK (ERK); activated ERK translocates to the nucleus and phosphorylates transcription factors that regulate gene expression

  58. A constitutively overactive mutant form of Ras is present in cells from a breast biopsy sample. Therefore, Ras in these cells

    • Overstimulates the MAP kinase cascade causing abnormal growth.