hormones lecture h04 h05

HormonesInsulin (RTK) Signaling

Dr. Aga Syed SameerCSIR Lecturer (Demonstrator)Department of Biochemistry,Medical College,Sher-I-Kashmir Institute of Medical Sciences, Bemina, Srinagar, Kashmir, 190010. India.

First MBBS

Lecture No: H 04

Time : 10:00am

Dated: 12/03/2015

• Insulin:

Insulin is anabolic hormone favoring synthesis of glycogen, TGAs and Protein

Insulin has a half life of 6 minutesProduced by β cells of Islets of Langerhans

Degraded by Insulinase (Liver)

• Insulin:

Insulin is dipeptide – one α Chain and One β Chainα Chain has 21 aas

β Chain has 30 aas

Two Disulfide linkages hold them together, with one extra in α Chain 6-11 with in α Chain

7-7 and 20-19 with α Chain and β Chain

Receptor Tyrosine Kinases

• Insulin: Insulin is antagonist to Glucagon in its function

Both hormones however respond to changes in blood glucose levels

↑ levels of Blood Glucose cause β-cells of pancreas to secrete Insulin

It functions as extracellular messenger molecule; informing cells that blood glucose levels are higher

Cells respond to Insulin by increasing Glucose uptake from the blood

Additionally, Insulin also causes its effects on metabolism as:↑ Glycogen Synthesis: Activating Glycogen Synthase Via GSKβ1

↑ Triglyceride Synthesis

↓ Gluconeogenesis: Deactivating Glycogen Phosphorylase


• Insulin Receptor (IR):

• Is hetero-tetramer –

Two α & two β subunits

• α-Chain:

Extracellular: Insulin Binding Site

• β-Chain:

Extracellular : Bound to α-Chain

Single Trans-membrane part

Cytoplasmic Part: Tyrosine Kinase Domain

Insulin receptor half-life is only 7 hours


• Single molecule of Insulin binds to Insulin Receptor for activation

• Binding causes Dimerization of IR by repositioning of the ligand binding domains of the α-chain on the outside of cell

• Then Tyrosine Kinase Domain of the Cytoplasmic Part of β-Chains come into close proximity (Juxtapositioning)

• This results in Trans-Autophosphorylation of the both β-Chains at several critical tyrosine residues;

• Thereby activating IR.

Insulin Receptor (IR)

• Phosphorylation occurs critically in the three tyrosine residues of Activation Loop of β-Chain.

• Important “Tyrosine” of RT is 960. Until and unless its phosphorylation occurs no signal cascade is created downstream

• Phosphotyrosine residues in turn act as Docking sites for the downstream proteins of the signal cascade

• The Phosphorylation causes conformational change in Activation Loop of β-Chain leaving catalytic cleft open for substrate binding.


• Activated “IR” then associates itself with Intracellular Effector Proteins - “Insulin Receptor Substrates” (IRSs) : IRS1 & IRS2

• IRSs have three Domains:• PH domain - N Terminal

• PTB Domain

• Tail containing Tyrosine Phosphorylation Sites

• The IRSs bind to IR via its Phosphotyrosine Binding (PTB) Domain

• IRSs on activation provide docking sites for all SH2-Domain Containing signaling proteins: PI3 Kinase, Grb2, PDK1

• The activated IRSs then interact with Phospholipids present on the inside of the Plasma Membrane


• PI3 Kinase has two subunits:• One contains two SH2 domains with which it binds to

Phosphorylation sites of Inositol of Phosphatidyl Inositol Other contains Catalytic site which causes phosphorylation of bound Inositol.

• PI3 Kinase is activated on binding with the PH Domain of Activated IRS1/2

• It function to phosphorylate the membrane bound PhosphatidylInositol Phosphates (at 3’ position of sugar) resulting finally in the production of PI-(3,4,5)P3

• PI-(3,4,5)P3 in turn mediates the function of Insulin signaling in regulating the blood Glucose Levels.

PI3Kinase (PI3K)

PI3Kinase (PI3K)

PI 3 Kinase products

(relevant to insulinsignallingis the production of PI-3,4,5-P3)

• Increased concentration of PIP3 recruits Protein Kinase B (PKB/Akt) to the membrane of the cell

• PKB is then phosphorylated by two membrane associated kinases PhosphoInositol Dependent Kinases (PDK1) and/or PKC

• Activated PKB is then released into the cytosol

• Causes phosphorylation of many proteins at Ser/Thr residues

• Helps in the translocation and fusion of GLUT4 containing vesicles with the plasma membrane via Rab4 protein

• GLUT4 then transports the Glucose from the blood into the cell

PI3Kinase (PI3K)

Protein Kinase B (PKB)

Rab4

PKB & GSK

AssignmentPatho-physiology of Diabetes Type IIInsulin and GlucagonHypo and HyperglycaemiaTypes of HypoglycaemiaInsulin Secretion and Signaling in Hypo/Hyperglycaemia

Effects of InsulinCross talk with other Signals – cAMP; Calcium; Leptin; Management of TD II

HormonesCalcium Signaling

Dr. Aga Syed SameerCSIR Lecturer (Demonstrator)Department of Biochemistry,Medical College,Sher-I-Kashmir Institute of Medical Sciences, Bemina, Srinagar, Kashmir, 190010. India.

First MBBS

Lecture No: H 05

Time : 10:00am

Dated: 12/03/2015

Calcium

• Extracellular calcium concentration is 5mM/L

• Intracellular free/ionized calcium concentration is very low ~0.05-10.0 uM/L

• Substantial amounts of calcium are associated with intracellular organelles such as Endoplasmic Reticulum & Mitochondria

• Inspite of this large concentration gradient & a favorable trans-membrane electrical gradient ; calcium is restrictedfrom entering the cell

Calcium Homeostasis

• Four calcium channels (mainly) play a critical role in regulating the calcium levels in the cytosol

• Two are highly functional in basal conditions

• Na+/Ca2+ channel in plasma membrane

• SERCA in Smooth Endoplasmic Reticulum

• Two are activated and in use under hormone or neurotransmitter action

• Voltage dependent Ca2+ channels in Plasma Membrane

• Ryanodine Receptors in Smooth Endoplasmic Reticulum

• There are three ways of changing the cytosolic calcium concentration:

By activating the receptors that serves as the channels (ligandgated Ca2+ channels) in the plasma membrane of the cell by hormone signaling.

Indirectly promoting the calcium influx by modulating the membrane potential at the plasma membrane, inturn opens the voltage gated Ca2+ channels.

Ca2+ can be mobilized from ER and also from mitochondrial parts

• Hormone action via Ca2+ involves the mediation of intracellular targets of Ca2+ ; which are Ca2+ - dependent regulator of “phosphodi-esterase /kinase activity”

Calcium Homeostasis

Calcium Homeostasis

Calmodulin – The Mediator• Is a Ca2+ - dependent regulatory protein

• 17kDA protein: Homologous to muscle protein troponin C in structure & function

• It has four Ca2+ - binding sites & full occupancy of these sites leads to a marked conformational change

• This allows calmodulin to activate Enzymes & Ion channels

• The interaction of Ca2+ with calmodulin is similar to the binding of cAMP to PKA and the subsequent activation of this molecule

• Calmodulin mediates its action via CaMKinase

Calmodulin

• Calmodulin is particularly involved in regulating various Kinases and Enzymes of cyclic nucleotide generation & degradation

• In addition, Ca2+/Calmodulin regulates the activity of many structural elements in cells:

• Actin-Myosin complex of smooth muscles

• Micro-filament mediated processes

• Mitosis

• Granule release

• Endocytosis

Calmodulin

• Number of critical metabolic enzymes are regulated by Ca2+, Phosphorylation /Both:

Glycogen Synthase;

Pyruvate Kinase;

Pyruvate Carboxylase;

Pyruvate Dehydrogenase;

Glycerol 3 Phosphate Dehydrogenase

Signaling• Hormone/Ligand activates GPCRs and Gq which inturn

activates PLCβ1

• IP3 and DAG activate Protein Kinase C

• PKC activity is inturn affected by calcium concentration in cytosol

• IP3 interacts with specific receptors (Ryanodine Receptors) and release calcium from SER into cytoplasm

• The activation of PKC and Increased calcium concentration activates “Ca2+ - Calmodulin Dependent Kinases (CaMK)” which mediates its effect

Ca2+/Calmodulin

Ca2+ Signaling in Synapse

Questions?