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 -ADRENERGIC  2 -ADRENERGIC GLUCAGON OPIATES

ADENYLATE CYCLASE.  -ADRENERGIC  2 -ADRENERGIC GLUCAGON OPIATES ACTH CHOLINERGIC ADENOSINE(A 2 ) ANGIOTENSIN PROSTAGLANDINS TSH. Ra. Ri. R CONTAINS HORMONE BINDING SITE ON EXTRACELLULAR FACE. MAY BE

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 -ADRENERGIC  2 -ADRENERGIC GLUCAGON OPIATES

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  1. ADENYLATE CYCLASE -ADRENERGIC 2-ADRENERGIC GLUCAGON OPIATES ACTH CHOLINERGIC ADENOSINE(A2) ANGIOTENSIN PROSTAGLANDINS TSH Ra Ri R CONTAINS HORMONE BINDING SITE ON EXTRACELLULAR FACE. MAY BE STIMULATORY OR INHIBITORY RECEPTORS C CATALYTIC PROTEIN. PREFERENTIAL REACTION WITH MnATP. Mr 190,000. NOT STIMULATED BY HORMONES, GTP OR F-. Gs GUANINE NUCLEOTIDE BINDING PROTEIN. CONFERS ON C THE ABILITY TO USE MgATP. ACTIVATES BY INCREASING AFFINITY FOR Mg2+ .  GsMr 45 ,000. HAS BINDING SITES FOR GTP. GTPase. SUBSTRATE FOR CHOLERA TOXIN. Gs Mr 35,000. REGULATORY PROTEIN TIGHTLY ASSOCIATED WITH Gs. Gs. Mr 8,000. PRENYLATED. ASSOCIATED WITH MEMBRANE. Gi GUANINE NUCLEOTIDE BINDING PROTEIN. INHIBITORY. SUBUNITS - Gi Mr 41,000. HAS BINDING SITES FOR GTP. GTPase. SUBSTRATE FOR BORTEDELLA PERTUSSIS TOXIN. GiMr 35,000. REGULATORY Gi.Mr 8,000. GsC ACTIVE WITH MgATP IN PRESENCE OF Gpp(NH)p (GUANOSINE 5' ,-IMIDO- TRIPHOSPHATE) OR GTP-S, CHOLERA TOXIN OR FLUORIDE. ACase g g b b a a Gs Gi

  2. STRUCTURE OF ADENYLATE CYCLASE (9 CLONED ISOFORMS) FIRST TRANSMEMBRANE CLUSTER SECOND TRANSMEMBRANE CLUSTER M1 M2 1 2 3 4 5 6 7 8 9 10 11 12 C1a C1b N bg-SYNERGISING SITE (AC11) C2a CaM REGULATORY REGION (AC1) CaM KINASE 11 REGULATORY REGION (AC111) P SITE (ATP BINDING SITE) FORSKOLIN BINDING SITES C C2b SIMILAR TO GUANYLATE CYCLASE

  3. Ca2+ Rp Ra Ra Ra Ri Ri Ri aq aq aq bg bg bg bg bg bg bg bg bg ai ai ai Ca2+ AC1,AC111 ACV111 ACV,ACV1 AC11,AC1V ACV11 CaM + + + + + + + IP3 PLC PLC PLC PKC PKC as as as _ _ _ _ _ Rp Ca2+ PKA REGULATION OF ADENYLATE CYCLASE Rp Ca2+

  4. Gas Gai Gbg FSK Ca2+ PK AC1 CaM- OR   CaM PKC FSK CaMK IVCaMK IV AC2   (WHEN  PKC STIMULATED BY GaS) AC3    CaM PKC CaMK II CaMK II AC4   PKC AC5  (b1g2)  PKA PKCa/ AC6  (b1g2)    PKA,PKC AC7   PKC AC8 Ca2+  CaM PKC AC9   CALCINEURIN

  5. STRUCTURE OF ADENYLATE CYCLASE Ca2+/CaM KINASE II (C2b) (INHIBITION) Ca2+/CaM KINASE IV (INHIBITION) PKA (INHIBITION)

  6. COMPARISON BETWEEN THE STRUCTURE OF ADENYLATE AND GUANYLATE CYCLASE

  7. STRUCTURE OF THE b-ADRENERGIC RECEPTOR T R D L K V F L R L I F A A E F Q K Q H E K S S S S S S S S S S E L C L R Q Q A K F K T T T 22-24 aas 402-477 AMINO ACIDS

  8. EXTRA CELLULAR e1 e4 SS e3 OH H2N OH OH tm1 tm4 tm2 tm3 INTRA CELLULAR i1 i2 STRUCTURE OF THE b-ADRENERGIC RECEPTOR

  9. DESENSITIZATION OF RECEPTORS PROCESS THROUGH WHICH PROLONGED EXPOSURE OF TARGET CELLS TO DRUG, HORMONE OR NEUROTRANSMITTER RESULTS IN DIMINISHED CELLULAR RESPONSIVENESS TO FURTHER AGONIST HETEROLOGOUS – FEEDBACK VIA CYCLIC AMP DEPENDENT PROTEIN KINASE. RATE OF PHOSPHORYLATION ENHANCED BY RECEPTOR OCCUPANCY. RESULTS IN FUNCTIONAL UNCOUPLING OF RECEPTOR FROM ADENYLATE CYCLASE. HOMOLOGOUS – HORMONE DESENSITIZES RECEPTOR TO ITSELF E.G. ADRENALINE AND THE b-ADRENERGIC RECEPTOR. NOT CYCLIC AMP DEPENDENT BUT VIA CYCLIC AMP INDEPENDENT KINASES – G PROTEIN COUPLED RECEPTOR KINASES E.G. b-ADRENERGIC RECEPTOR KINASE bARK

  10. DESENSITIZATION OF RECEPTORS N N G PROTEIN EFFECTOR ACTIVATION ACTIVATED G PROTEIN ACTIVATION BASAL Gs N b-AGONIST C C C bARK b-ARRESTIN N N b-ARRESTIN P P P P P P P P C C b-ARRESTIN

  11. BASAL N C P P P P P P P P P P P P b-ARRESTIN b-ARRESTIN DESENSITIZATION OF RECEPTORS HORMONE N N C C bARK b-ARRESTIN INTERNALISATION (CLATHRIN BINDING) RECYCLING b-ARRESTIN N N PHOSPHATASE C C Pi

  12. DESENSITIZATION OF RECEPTORS T1/2% OF TYPE RECEPTOR LOST bARK 0.1 -1 50-70 HOMOLOGOUS* PKA 1 –3 20-50 HETEROLOGOUS ** SEQUESTRATION 1-60 10-60 HOMOLOGOUS DOWN REGULATION 0.5-24hr 20 HOMOLOGOUS/ HETEROLOGOUS mRNA 0.5-24hr 20 HETEROLOGOUS STABILIZATION * HIGH AGONIST CONCENTRATIONS ** LOW AGONIST CONCENTRATIONS

  13. g a b GDP g a b GDP GTP Pi g a + b GTP EFFECTOR EFFECTOR G PROTEIN CYCLE AGONIST-LIGANDED RECEPTOR g SUBUNITS PRENYLATED (FARNESYL OR GERANYL GROUPS) C TERMINUS OF a SUBUNIT Ras-LIKE. N TERMINUS HELICAL. IN SOME CLASSES a SUBUNIT MYRISTOYLATED OR PALMITOYLATED AMPHIPATHIC a HELICES OF a, b AND g SUBUNITS 20a SUBUNITS 5 b SUBUNITS 10+ g SUBUNITS as ACTIVATES ACase ai INHIBITS ACase aq ACTIVATES PLCb

  14. COMPARISON BETWEEN GSa AND Ras

  15. P P P THE G PROTEIN bg CYCLE R bARK a-GTP g b RGS (GAPS) a GDP a-GDP R R g b g b bARK bARK

  16. b c e g f f b g d a c b d a g e a d e g bARK c b f COILED COIL STRUCTURE OF bARK AND bg SUBUNITS

  17. EFFECTORS REGULATED BY G PROTEIN SUBUNITS a SUBUNIT bg SUBUNIT ADENYLATE CYCLASE 1 ADENYLATE CYCLASE 1 ADENYLATE CYCLASE 11(1V) ADENYLATE CYCLASE 11(1V) ADENYLATE CYCLASE 111 ADENYLATE CYCLASE 111 PHOSPHOLIPASE Cb1 PHOSPHOLIPASE Cb1 PHOSPHOLIPASE Cb2 PHOSPHOLIPASE Cb2 PHOSPHOLIPASE Cb3 PHOSPHOLIPASE Cb3 K+ CHANNEL (IKAch) K+ CHANNEL K+ CHANNEL (IKATP) – _ RECEPTOR KINASES cGMP PHOSPHODIESTERASE – _ PHOSPHOLIPASE A2 CALCIUM CHANNEL CALCIUM CHANNEL(?)

  18. cAMP DEPENDENT PROTEIN KINASE • COMPOSED OF 2 REGULATORY AND 2 CATALYTIC SUBUNITS. • THE FUNCTION OF THE REGULATORY SUBUNIT IS BIND TO AND INHIBIT THE CATALYTIC SUBUNIT • R2C2 + 4cAMP R2(cAMP)4 + 2C • (Inactive) (Active) • AFFINITY OF REGULATORY SUBUNIT 0.02-0.06nM (DECREASED 10,000X IN PRESENCE OF cAMP • 2 REGULATORY SUBUNITS – TYPE 1 (47kDa) AND 11 (54 kDa) • TYPE 1 DISSOCIATED IN HIGH SALT AND HIGH HISTONE • TYPE 1 REASSOCIATES SLOWLY, TYPE 11 FAST • TYPE 11 UNDERGOES PHOSPHORYLATION/DEPHOSPHORYLATION • AUTOCATALYTIC REACTION INCREASING THE SENSITIVITY TO cAMP AND AFFINITY OF REGULATORY SUBUNIT FOR CATALYTIC. • TYPE 11 HAS ARG-ARG-X-SER WHEREAS TYPE 1 ARG-ARG-X-ALA • INHIBITED BY HEAT STABLE INHIBITOR, 75 AMINO ACIDS WITH PSEUDO-SUBSTRATE DOMAIN

  19. TYPE 1 AND 11 cAMP-DEPENDENT PROTEIN KINASE R 50-55kDa C 40kDa ARG-ARG-X-SER COOH C C R H2N C C NH2 HOOC R cAMP INHIBITOR PROTEIN + HINGE REGION + AUTOINHIBITORY REGION PSEUDOPHOSPHORYLATION SITE AUTOPHOSPHORYLATION OF TYPE 11 INCREASES AFFINITY FOR cAMP

  20. KINASE KINASE RII P ANCHORING PROTEIN LOCALISATION OF KINASES AND PHOSPHATASES BY ANCHORING PROTEINS SUBSTRATE TARGETING LOCUS

  21. PHOSPHODIESTERASES 330 RESIDUES N C TARGETTING PDE4 SH3 BINDING DOMAIN PDE3 REGULATORY PDE1 – CaM INHIBITORY PDE2 – cGMP STIMULATORY PDE3 – PKA & INSULIN STIMULATED KINASE PDE4 - PKA CATALYTIC REGULATORY PDE1 1. Ca2+ /CALMODULIN-DEPENDENT PDE MONOMER 58-75 kDa. N-TERMINAL CALMODULIN BINDING DOMAIN. SEVERAL FORMS SHOWING DIFFERENT ACTIVITIES TOWARDS cGMP AND cAMP. REVERSIBLY ACTIVATED BY CALMODULIN IN THE PRESENCE OF Ca2+ (Ca2+ )4 (Km , Vmax). PDE2 + CAL2 +4Ca2+ PDE2.CAL2 PHOSPHORYLATED BYcAMPPK CAUSING DECREASED AFFINITY OF CALMODULIN FOR ENZYME AND ACTIVATION BY Ca2+/CALMODULIN.

  22. PHOSPHODIESTERASES  2. cGMP STIMULATED PDE DIMER 105kDa. TWO cGMP BINDING SITES ON REGULATORY DOMAIN. cGMP ENHANCES ACTIVITY. Km FOR cAMP - ENHANCED BREAKDOWN.  3. cGMP INHIBITED PDE MONOMER 105-135 kDa. PREFERRED SUBSTRATE cAMP. cGMP ACTS AS COMPETITIVE INHIBITOR OF cAMP HYDROLYSIS. ACTIVATED BY PHOSPHORYLATION BY THE cAMPPK. ACTIVATED BY PHOSPHORYL- ATION BY THE INSULIN- SENSITIVE PROTEIN KINASE. 4. cAMP SPECIFIC PDE MONOMER 60-80kDa. INDUCED BY cAMP. NO KNOWN SHORT TERM MODULATORS. 5. cGMP SPECIFIC PDE DIMER. 90-95kDa. 2cGMP BINDING SITES IN ADDITION TO ACTIVE SITE. INVOLVED IN RHODOPSIN-TRANSDUCIN PATHWAY.

  23. PROTEIN PHOSPHATASES SERINE/THREONINE PHOSPHATASE TYPE 1 CONTAIN A CATALYTIC SUBUNIT INHIBITED BY TWO HEAT STABLE INHIBITORS, INHIBITOR-1 AND INHIBITOR-2. PREFERENTIALLY DEPHOSPHORYLATES b-SUBUNIT OF PHOSPHORYLASE KINASE. CATALYTIC SUBUNIT PRESENT AS PART OF A LARGER COMPLEX, 90% HOMOLOGY BETWEEN FORMS. REGULATORY SUBUNIT DIFFERS. ASSOCIATED WITH GLYCOGEN PARTICLES, MICROSOMES AND RIBOSOMES. ACTS ON ENZYMES OF GLYCOGEN METABOLISM, PROTEIN SYNTHESIS AND CHOLESTEROL METABOLISM. GLYCOGEN/SARCOPLASMIC RETICULUM FORM – COMPLEX OF 37kDa CATALYTIC SUBUNIT AND 103kDa GLYCOGEN BINDING PARTICLE. PHOSPHORYLATION OF GLYCOGEN BINDING PARTICLE BY PKA CAUSES RELEASE OF PP-1. LEADS TO INTERACTION WITH PI-1 AND INHIBITION. PI-1 ACTIVE AFTER PHOSPHORYLATION BY PKA. ATP-Mg DEPENDENT FORM - COMPLEXES WITH PI-2 TO KEEP INACTIVE. ACTIVATION OCCURS BY PHOSPHORYLATION OF PI-2 BY GLYCOGEN SYNTHASE KINASE 3. CAN AUTODEPHOSPHORYLATE ITSELF.

  24. PROTEIN PHOSPHATASES TYPE 2 INSENSITIVE TO INHIBITOR PROTEINS. PREFERENTIALLY DEPHOSPHORYLATES a-SUBUNIT OF PHOSPHORYLASE KINASE. TYPE 2A (PP-2A) 36kDa C SUBUNIT, 60kDa A SUBUNIT, 54kDa B SUBUNIT. ACTIVATED BY BASIC PROTEINS (e.g. HISTONES, SPERMINE). ACTIVE TOWARDS ENZYMES OF GLYCOLYSIS, FATTY ACID SYNTHESIS, GLUCOENOGENESIS, AROMATIC AMINO ACID DEGRADATION. MAJOR PHOSPHORYLASE KINASE a-SUBUNIT PHOSPHATASE IN ABSENCE Ca2+. TYPE 2C (PP-2C) DIMER OF 44kDa SUBUNIT. ACTIVE TOWARDS CHOLESTEROL BIOSYNTHESIS, MYOSIN LIGHT CHAINS, CASEIN. BROAD SPECTRUM (PDH PHOSPHATASE FAMILY). TYPE 2B (PP-2B) CONSISTS OF A SUBUNIT (CATALYTIC) AND B SUBUNIT (CALCIUM BINDING). DEPHOSPHORYLATES INHIBITOR-1 AND REGULATORY SUBUNIT OF PKA, a-SUBUNIT OF PHOSPHORYLASE KINASE AND MYOSIN LIGHT CHAIN KINASE. BINDS TO CALMODULIN TO INCREASE ACTIVITY 10-FOLD. INCREASES Vmax. LITTLE ACTIVITY TOWARDS METABOLIC ENZYMES

  25. _ _ PROTEIN P P P P P cAMP PATHWAY cAMP AC1 + 2Cal + 4Ca2+ PDE3 PDE3 INSULIN AC1.Cal 2.(Ca2+)4 METHYL XANTHINES PDE1 + 2Cal + 4Ca2+ ATP HORMONE GTP 5'AMP AC PDE1.Cal 2.(Ca2+)4 cAMP R2C2 (Inactive) R2(cAMP)4 2C 2I PROTEIN 2CI (Inactive) + PPI PI-1 PI-1 PPI.PI-1 (Inactive) GSK3 P2B.Cal 2.(Ca2+)4 PI-2 (Inactive) PPI.PI-2 (Inactive) P2B + 2Cal + 4Ca2+ (Inactive)

  26. INTERACTION BETWEEN cAMP and Ca2+ cAMP PHOSPHORYLATION TYPE II R SUBUNITS PK ACTIVITY PHOSPHORYLATION PI-1 PP-1 ACTIVITY PHOSPHORYLATION Ca 2+/CALMODULIN PDE  cAMP BREAKDOWN PHOSPHORYLATION RECEPTORS ADENYLATE CYCLASE ACTIVITY * PHOSPHORYLATION cGMP INHIBITED PDE cAMP BREAKDOWN ENHANCED INSULIN EFFECT* INDUCTION cAMP SPECIFIC PDE* Ca2+ PP2B PHOSPHORYLATION TYPE II R SUBUNITS PK ACTIVITY  PHOSPHORYLATION OF PI-1, PP-1ACTIVITY Ca2+/CALMODULIN PDE cAMP BREAKDOWN EMPHASISES Ca2+ RESPONSE, DE-EMPHASISES cAMP *LONG TERM RESPONSE

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