hormones lecture h02

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  1. 1. Hormones G Proteins Signaling Dr. Aga Syed Sameer CSIR Lecturer (Demonstrator) Department of Biochemistry, Medical College, Sher-I-Kashmir Institute of Medical Sciences, Bemina, Srinagar, Kashmir, 190010. India. First MBBS Lecture No: H 02 Time : 10:00am Dated: 07/03/2015
  2. 2. The 2012 Nobel Prize in Chemistry was awarded jointly to Robert J. Lefkowitz (l) and Brian K. Kobilka for studies of G-protein-coupled receptors G Protein Coupled Receptors (GPCRs)
  3. 3. They are the receptor named just because they interact with G Proteins. Also referred to as Serpentine Receptors as they contain seven trans-membrane helices (7TM) Largest known receptor family Constitutes > 1% of the human genome. Comprises receptors for a diverse array of molecules: neurotransmitters, odorants, lipids, neuropeptides, large glycoprotein hormones G Protein Coupled Receptors (GPCRs)
  4. 4. Their Structure consists of: Amino- Terminal: Present on the outside of the cell Seven helices: traversing the plasma membrane & connected by loops at varying length Carboxyl-Terminal: Present on Inside of the cell Ligand binding site: three loops that are on outer surface of the cell Docking site: three loops that are on inner surface/ cytoplasmic side of the cell; provide site for binding of intracellular protein G proteins G Protein Coupled Receptors (GPCRs)
  5. 5. G Protein Coupled Receptors (GPCRs)
  6. 6. They are named so because they bind to guanine nucleotide as prosthetic group. GDP Inactive form GTP Active form Hetero-trimeric in nature : Three different polypeptide subunits , & . Held at plasma membrane by lipid chains that are covalently attached to the & . G Protein
  7. 7. The guanine nucleotide binding site is present on G subunit: In GDP bound conformation (Inactive, b): G subunit has high affinity for the G; hence they remain together as trimer on cell surface In GTP bound conformation (Active, a): : G subunit has low affinity for the G; leading to its dissociation from complex The two conformations are inter-convertible via activation by GPCRs; which cause GDP-GTP switching on G subunit of trimer. Each dissociated G subunit in turn is free to activate an effector protein Like Adenylyl Cyclase etc. G Protein
  8. 8. G Protein
  9. 9. Thus, G subunit is said to be On when it is bound to GTP. G subunit turn itself Off by hydrolysis of bound GTP via its intrinsic GTPase activity. The G subunit is categorized into four types: Gs: Stimulatory: cAMP levels; Adenylyl cyclase activity; Cardiac Ca2+ Gi: Inhibitory: cAMP levels ; Adenylyl cyclase activity; K+ Channels Gq: Stimulatory: Phopholipase C- B1; IP3; PIK3 G12/13: Unknown: Cl- Channels GEF: Guanosine Exchange factors: cause GTP GDP switching GAP: GTPase activating Protein: Increases the rate of GTP hydrolysis RGS: Regulators of G protein Signaling: Increases the rate of GTP hydrolysis G Protein
  10. 10. G Protein
  11. 11. The G Protein Cycle
  12. 12. The G Protein Cascade
  13. 13. Cyclic AMP Cyclic GMP Inositol Tri Phosphate - IP3 Diacyl glycerol - DAG Ca/Calmodulin Smad Second Messengers
  14. 14. Are small easily diffusible chemicals/intermediates Have very short half life They relay signals received at the receptors on the cell surface Serve to enhance the strength of the signal Affect more than one pathway and/ or protein Cause divergence of the signal Second Messengers
  15. 15. Cyclic AMP Cyclic GMP Ca2+ Diacylgycerol Protein substrates PK-A PK-G Calmodulin PK-C Protein Ser/Thr kinases Protein substrates Protein substrates Protein substratesMultifunctional kinases Other phospholipases 1 2 3 4 5 1 2 3 4 5 Tyrosine kinase IP3 G G G G InsulinGlucagon T-cell Activation Nitric oxide G protein End result is phosphorylation of one or more proteins
  16. 16. Benefits of a 2 messenger system Amplification Signal molecule Receptor protein Activated adenylyl cyclase Amplification Amplification Amplification Amplification GTP G protein 2 1 3 4 5 6 7 Enzymatic product Enzyme Protein kinase cAMP Not yet activated
  17. 17. Produced by activation of Adenylyl Cyclase by G from ATP. Has capability to easily diffuse to other sites within the cell. cAMP molecules diffuse into cytoplasm and bind to regulatory subunits of cAMP dependent Protein Kinase A Protein Kinase A is its effector protein via which cAMP mediates its function cAMP
  18. 18. Cyclic AMP System Receptor Adenylate cyclase G-protein Protein Kinase A c-AMP Stimulate (Gs) and Inhibit (Gi)
  19. 19. cAMP
  20. 20. cAMPisgeneratedfromATP byanenzyme:adenylyl cyclase. ACisregulatedbyGproteins cAMP
  21. 21. cAMP activates one or more kinases.
  22. 22. Is a hetero-tetramer Two regulatory & two catalytic subunits (R2C2). The regulatory subunits normally inhibit the catalytic activity of enzyme. The cAMP binding to regulatory subunits cause dissociation of regulatory subunits from the tetramer Therefore, results in release of the catalytic subunits of PKA for further downstream function. Protein Kinase A - PKA
  23. 23. PKA either function to regulate the metabolism by phosphorylating the key metabolic enzymes like: Glycogen Phosphorylase, Gylcogen Synthase etc. Activates Glycogen Phosphorylase Used in utilisation of glycogen, degrades it to release glucose in blood stream Inactivates Glycogen Synthase Used in assimilation of glucose to glycogen, uptakes glucose from blood stream and stores in liver & muscles as glycogen Activates Phosphorylase Kinase Protein Kinase A - PKA
  24. 24. Or some of it translocates to the nucleus In nucleus it phosphorylates key nuclear proteins which function as transcription factor called CREB (cAMP Response element Binding- Protein) CREB forms dimer and then binds to the DNA at particular sequences within the promoter/regulatory region 5- TGACGTCA known as CRE (cAMP Response Element) Protein Kinase A - PKA
  25. 25. Protein Kinase A & Its Effects
  26. 26. Cholera toxin catalyzes covalent modification of Gs. ADP-ribose is transferred from NAD+ to an arginine residue at the GTPase active site of Gs. ADP-ribosylation prevents GTP hydrolysis by Gs. The stimulatory G-protein is permanently activated. Pertussis toxin (whooping cough disease) catalyzes ADP-ribosylation at a cysteine residue of the inhibitory Gi, making it incapable of exchanging GDP for GTP. The inhibitory pathway is blocked. ADP-ribosylation is a general mechanism by which activity of many proteins is regulated, in eukaryotes (including mammals) as well as in prokaryotes. Toxic Effects
  27. 27. Questions