BioC 325: Lecture 4: Effector Systems and Second Messengers PDF

Effector Systems and Second Messengers Downstream of GPCRs (Part1) 1  Define the important effector systems and the related second messengers downstream of Gα Appreciate the role of these systems in the subsequent processing of the...

Effector Systems and Second Messengers Downstream of GPCRs (Part1) 1  Define the important effector systems and the related second messengers downstream of Gα Appreciate the role of these systems in the subsequent processing of the extracellular signal 2  GPCR Downstream Signaling Cyclases: AC, GC Effector Phospholipases Phosphodiesterases (PDEs) βARKs RhoGEFs Ions (Ca2+, K+) Ion channels cAMP, cGMP Arachidonic acid Phosphoinositides 2nd Messenger Diacylglycerol Gases (NO, CO) AGC family: PKA, PKG, & PKC PKB/Akt CAMK: Ca2+/calmodulin Second messenger –dependent activated protein kinase Protein Ser/Thr Kinases 3  Relay molecules (effectors, kinases…) and second messengers have essentially the same jobs in signal transduction pathways: passing-on the signal intracellularly.  However, relay molecules are almost always proteins that require activation. They are large and do not diffuse through the cell quickly.  On the other hand, second messengers are small, water soluble, easily diffusible non-protein molecules that allosterically activate the next relay proteins  cAMP and Ca2+ are common second messengers in human systems. 4 Effector Enzymes 2nd messenger- Coupled to Associated dependent GTP-binding 2nd messenger Ser/Thr Proteins kinase Adenylyl cyclase cAMP PKA (AC) Phospholipase C IP3, DAG PKC (PLC) 5  AC-cAMP-PKA pathway  PLC-(IP3 & DAG)-PKC pathway 6  AC-cAMP-PKA pathway  PLC-(IP3 & DAG)-PKC pathway 7 cAMP: cyclic adenosine monophosphate 8 Regulation of AC Activity 9  9 membrane-bound isoforms: all are activated by Gαs  Type 1 was first purified from bovine brain  Gαs : activates all the AC isoforms in the GTP-bound form  All isoforms (except AC9) are activated by forskolin (Fsk) 10 11  Sequence similarities = 50 % at the amino acid level  Structure and the TM domains resemble those of transporters although only enzymatic activity has been described to date for these proteins  93 % conserved in the C1a and the C2a domains 12  Three main classes: 1. AC 2, 4 and 7: synergistically activated by Gαs and Gβγ subunits 2. AC 5 and 6: inhibited by Gαi and Ca2+ (*Calmodulin (CAM)-independent pathway) 3. AC 1 and 8 (and probably 3): synergistically activated by Gαs and Ca2+-CAM *Calmodulin: calcium binding protein 13  M1 M2  12 α-helical TM Potential N- domains: glycosylation ◦ 6 helices at M1 sites ◦ 6 helices at M2  2 similar catalytic domains: C1 and C2 each having two subdomains: a & b Short Cytoplasmic Cytoplasmic N-terminus C-Terminus  Both C- and N- terminals are AC in its inactive conformation: cytoplasmic C1a and C2a domains are apart 14  Fsk is a diterpene that is produced by the Indian Coleus plant and is commonly used to raise levels of cyclic AMP in the study and research of cell physiology ◦ Forskolin (Fsk): strong activator of AC  Has positive inotropic effect: increases heart rate and lowers blood pressure  GI effects: non-specific spasmolytic activity  CNS effect: depressent (at large doses)  Fsk: hydrophobic activator  gluing together the two domains of the active core ( Head to tail fashion)  GTP-Gαs gluing as Fsk 15 Activation ATP The two domains Fsk C1a and C2a contact each other to form Gαs binding sites for ATP, fsk, Gαs and Gαi AC in its active conformation: Dimer formation between C1a and C2a 16 Head to Tail Fashion Cooper DM and Crossthwaite AJ (2006) Higher-order organization and regulation of adenylyl cyclases. Trends Pharmacol Sci 27(8):426-431. 17  The C1 and C2 domains are together sufficient to catalyze the conversion of ATP to cAMP but with a very low activity. However their association rate and activity is enhanced100-folds by Fsk or Gαs. Fsk and Gαs: These two activators are synergistic, the presence of the one enhancing the affinity of the other. 18  19 Tesmer et al., 1999 Two-metal-Ion catalysis in adenylyl cyclase. Science 285(5428):756-760.  Gαs facilitates closure of the active site Gαi stabilizes a around ATP more open inactive Fsk facilitates conformation In AC2: Gβγ binds to C2a and facilitates closure of the conformational changes active site Substrate to cooperatively stimulate around ATP the enzyme 20  C2a Cytosol Plasma membrane Extracellular 21  CAM associates with the C1b : regulation of the Ca2+ - sensitive AC (1 and 8 )  Gai = acts only on AC 1, 5 and 6  differrent binding sites than Gas  Gbg = activation of AC2 when Gas is bound 22  Many different cell responses are mediated by cAMP.  These include: ◦ increase in heart rate ◦ cortisol secretion ◦ breakdown of glycogen and fat  Uncontrolled cAMP-dependent pathway can ultimately lead to hyper-proliferation: development and/or progression of cancer. 23  Mainly PKA-mediated nonPKA-mediated actions actions Epac: Exchange cAMP-gated C Protein directly R ion channels Activated by R C cAMP PKA 24  Nomenclature: cAMP-dependent protein kinase. (Also called protein kinase A)  Function: phosphorylates proteins at the Ser/Thr residues 25  2 Catalytic subunits: ◦ 3 isoforms in each subunit: α, β, and γ ◦ Isoforms are tissue-specific  2 Regulatory subunits (RI and RII) ◦ Two major isoforms: I and II ◦ 35% homology in cAMP-binding domain (between RI and RII) ◦ Origin of RI subunits: skeletal muscles ◦ Origin of RII subunits: cardiac muscles ◦ α and β subtypes in each isoform 26  Structure-Function Relationship of PKA Blocked substrate- binding sites  An inactive stable tetramer denoted R C as C2R2 1. 2 catalytic subunits (2C) R C Inactive PKA 2. 2 regulatory subunits (2R) Activated  Here, substrate binding sites of C AC subunits are blocked by the autoinhibitory domains of the R subunits cAMP Once activated by cAMP binding: dissociation of tetramer: R C 1. cAMP-bound R dimer: 2R (autoinhibitory domains burried) R C 2. Active free catalytic monomers Active PKA Open substrate- (open substrate-binding sites) binding sites 27  Cytosolic actions: (short term regulation) ◦ Example: Regulation of glycogenolysis between meals  Nuclear actions: (long term regulation) ◦ Phosphorylation of transcription factors: CREB (cAMP response element binding protein)  gene expression ◦ Example: stimulation, by catecholamines, of the synthesis of mRNA coding for the β-adrenergic receptor 28 exercise or β2-adrenergic starvation receptor in the liver Gαs-GTP glycogen (inactive form) (Active form) 29  Following exercise or starvation, glycogen breakdown occurs in liver through the action of β2- adrenergic receptor (a GPCR coupled to Gαs)  AC hydrolyzes ATP to cAMP, which activates PKA.  Activated PKA phosphorylates two enzymes: 1. Phosphorylase kinase: Whose p~ form in turn phosphorylates phosphorylase b active p~phosphorylase a that breaks down glycogen to glucose-1-p 2. Glycogen Synthase a changing it to  inactive p~ glycogen synthase b, which stops the utilization of UDP-glucose for the synthesis of glycogen 30 Activated PKA: 1. Favors glycogen breakdown Net overall effect: enhanced 2. Blocks Glycogen Synthesis glycogenolysis 31 The net result: Signal Amplification Very weak first A large kinetic signal: signal: Low receptor Transduction Rapid occupancy by a Glycogenolysis hormone 32  Signal Amplification: Is mediated by two phosphorylation mechanisms: PKA 1st intermediate signal p~ amplification Phosphorylase kinase 2nd intermediate p~ signal amplification Phosphorylase a Each kinase enzyme can activate more than one of the next kinase enzymes in the sequence  Signal amplification 33 Signal Amplification by Phosphorylation: Importance of Kinases 34 35  Catalytic subunit of PKA can diffuse into the nucleus and phosphorylate CREB at the Ser residue.  This phosphorylation activates CREB.  The active CREB binds specifically at CRE (cAMP response element) in promoter region resulting in gene expression. 36 37  AKAP = A-kinase anchoring protein  Scaffold for the interaction of PKA with the receptor  Importance in Signaling: ◦ AKAP is needed for receptor phosphorylation by PKA ◦ Role in desensitization: decrease in the subsequent receptor signal 38  AKAP79 associates with β2-adrenergic receptors (β2-AR). This enhances β2-AR-induced cAMP-PKA signaling by recruiting PKA close to the receptor and the site of adenylyl cyclase activation. PKA phosphorylation of the β2-AR leads to desensitization of the receptor; however, PKA phosphorylation enhances glutamate receptor activity. Thus AKAP79 brings the cAMP- generating machinery, PKA, and the substrates into close proximity. In addition to anchor PKA, AKAP79 also recruits protein kinase C (PKC) and protein phosphatase 2B (PP2B) and thereby integrates several signalling pathways into a multiprotein complex. TASKÉN K , and AANDAHL E M Physiol Rev 2004;84:137-167 ©2004 by American Physiological Society  Phosphorylation of β2- adrenergic receptors by PKA diverts its attention from Gs to Gi signaling.  The resulting activation of βγ subunits (partners of Gi) initiates a pathway of reactions leading to the activation of ERK and subsequent nuclear signals 40 Nomenclature: Exchange Protein directly Activated by cAMP  Function: A guanine nucleotide exchange factor (GEF)  Structure: ◦ Has sequence homology to Ras-GEF and Rap1-GEF ◦ Has Cyclic-nucleotide binding domains for cAMP (cAMP binding domains)  2 isoforms = Epac1 and Epac 2 41  cAMP binding domains DEP, Dishevelled, Egl-10-Pleckstrin; REM, Ras-exchange motif; RA, Ras-associated domain; Yu, Z., and Jin, T. (2010). Cell. Signal. CDC25HD, CDX25-homology domain 22, 1-8 42 43 Epac- conserved cAMP-binding domain that acts as a molecular switch to sense intracellular cAMP levels in order to control diverse biological functions. The existence of two effectors of cAMP (EPAC and PKA) provides an instrument for a more specific and integrated control of cAMP signaling pathways in a spatial and temporal manner to modulate a specific cellular function. 44 Effector Systems and Second Messengers Downstream of GPCRs (Part2) PLC-(IP3 & DAG)-PKC pathway 45