cell signalling lecture

7/29/2019 cell signalling lecture

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Adenylyl cyclase


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Cell surface receptors

G-protein Linked Receptor

Receptor Protein Tyrosin Kinase

Cytokine receptors and Nonreceptor Protein- Tyrosin

Kinase

Receptors linked to other Enzymatic activities


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G-protein Linked Receptor

They consist of single polypeptide

chain which have seven membrane

spanning alpha helices

These make Several sensory

receptor including Olfactoryreceptors, rhodopsin etc


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The switch is turned off when G protein hydrolyses its own

bound GTP converting it back to GDP.

But during this period the active protein has opportunity to

diffuse away from the receptor and deliver message to its

own downstream target.


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Most G protein linked receptors activate a chain of events

that alter the concentration of one or more small

intracellular mediators.

Cyclic AMP and Ca2+ ions are most widely used

mediators.

Cyclic AMP pathwayCyclic AMP is synthesiszed from ATP by membrane bound

enzyme adenylyl cyclase.

It is rapidly destroyed by one or more cyclic AMP

phospodiesterases which hydrolyze cyclic AMP to

adenosine 5` AMP.

Cyclic AMP dependant protein kinases mediates the effect

of cyclic AMP


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Protein kinases catalyzes the transfer

of the terminal phosphate group from

ATP to specific serine or threonine of

selected proteins (enzymes)

Protein Kinase A consists of a

complex of two catalytic and two

regulator subunits.

The binding of cyclic AMP alters the

confirmation of of regulatory subunits,

causing them to dissociate fromcomplex.

The realease catalytic subunit are

thereby activated to phosphorylate

specific proteins.


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The activity of an protein regulated by phosphorylation

depends on the balance at any instant between the

activities of kinases that phosphorylates it andphosphatases that are constantly dephosphorylating it

In some animal cells cyclic AMP activates the transcription

of specific genes. The genes contains a regulatory subunitcalled cyclic AMP response elements or CRP.

It is phosphorylated by protein kinases leading to activation

of genes.


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Receptor protein tyrosine Kinase

Unliked G linked receptors either surface receptors aredirectly linked to intracellular enzyme.

Largest family of such receptor is the receptor protein tyrosin

Kinase which phosphorylates their substrate proteins on

tyrosine residues.

More than 50 receptors protein Tyrosine Kinases have been

identified including EGF, NGF, PDGF, insulin and many

others.

N terminal extracellular ligand binding domain , a single

transmembrane alpha helix and a cytosolic C terminal domain

with protein tyrosine Kinase activity


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Most of them are single polypeptide chain although insulin

receptor and some related receptor are dimers


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Ligand binding induces the dimerization of receptor

Ligand inducced dimerization then lead to

autophosphorylation of the receptors as dimerised

polypeptide chain cross phosphorylate one another.

This auto-phosphorylation plays two key roles first

phosphorylation of Tyrosine residue within the catalytic

domain may play a regulatory role by increasing receptorprotein kinase activity.

Binding of ligand the extracellular domain of these receptors

activates ther cytosolic kinase domains resulting in

phosphorylation of both the receptors themselves and

intracellular target proteins that propagate the signal initiated

by growth factor binding.


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Phosphorylation of Tyrosine residue of catalytic domain

creates specific binding sites for additional proteins that

permits intracellular signals downstream of the activatedreceptor.

The association of these downstream signaling molecules

with receptor protein tyrosin kinases is mediated by Protein

domain that bind to specific phosphotyrosine kinase

SH2 domain consists of approximately a hundred amino

acids and bind to specific short peptide sequences

containing phospho tyrosine residues.


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Cytokine receptors function in association with nonreceptor protein Tyrosine Kinase

Cytokine receptors function in association with non receptorprotein tyrosine kinase, which are activated as a result of

ligand binding.

Ligand binding induces receptor diamerization and crossphosphorylation of associated nonreceptor protein tyrosine

kinase .

These activated Kinases then phosphorylates the receptors,

providing phosphotyrosine binding sites for the recruitment ofdownstream signaling molecules that contain SH2 domain.

Non receptor protein Tyrosine Kinase associated with

cytokine receptors fall into two major catagories. Many of


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Other family is Janus Kinase or Jak Family. They are

universally required for signaling from cytokine receptors.


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Receptor linked to other enzymatic activities.

These receptors include protein tyrosine phosphatase,protein serine Thrionine Kinase and Guanyl cyclase.

Protein Tyrosine phospahatases removes phosphate group

from phosphotyrosine residues and counter balance the

effect of protein Tyrosine Kinase.

Receptors for transforming growth factors and related

polypeptides are protein Kinases that phosphorylates Serine

and Therionine on substrate protein.

TGF is a family of polypeptide growth factor that controls

proliferation and differentiation of various growth cells.


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Binding of ligand to these receptors result in the assocaition of

two distinct polypeptide chans which are encoded by the

family of TGF rceptor gene family to form hetrodimers in

which receptor Kinases cross phosphorylates one another.

Some polypeptide ligands bind to receptor whose cytosolic

domain are guanylyl cycalse which catalyze formation of GMP.

The receptor has extracellular ligand domain, a single

transmembrane alpha helix and cytosolic domain with

catalytic activity.

Ligand binding stimulates cyclase activity leading to formation

of GMP a second messenger.


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Calcium ion Signaling

Resting concentration of Ca2+ is kept low in cytosol and this is

achieved n several ways.

Two pathways of ca2+ signaling have been well defined . First

one is studied in nerve cells in which depolarization of Plasma

membrane causes an influx of ca2+ into the nerve terminal

initiating the secretion of neurotransmitters.

Calcium ions enter through voltage gated ion channels that

open when PM of nerve terminals is depolarized by an invading

action potential.

In second Ubiquitous pathway the binding of ligand to receptor

on surface causes release of ca2+ from Endoplasmic reticulum.

This is done through another intracellular messenger molecule

inositol.


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Phospholipids and Ca2+

Phospthatidylinositol 4-5 bisphosphate (PIP2). PIP2 is a minor

component of inner leaflet of Plasma membrane. Severalgrowth hormones and growth factors stimulate hydrolysis of

PIP2 by phospholipase C which produces two distinct second

messenger diacylglycerol and inositol1,4,5triphosphate (IP3)

Diacylglycerol and IP3 stimulates distinct downstream signaling

pathways (Protein KinaseC and Ca2+ ion mobilization

respectively)

PIP2 hydrolysis is activated by both G protein coupled receptors

and protein Tyrosine Kinase.


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It happens because one form of Phospholipas C is stimulated by G

protein coupled receptors while a second enzyme (PLC-) contains

SH2 domain that mediates its association with activated receptorprotein Tyrosine Kinase.

Diacylglycerol activates Protein Kinase C family protein many of

which play role in cell growth and differentiation.

A good example is phorol esters action . Tumor promoting activity

of it is base on its ability to stimulate Protein Kinase C by acting as

anologue of diacylgylcerol. Protein Kinase C then activates other

intracellular targets, including MAP kinase pathaway, leading totranscription factor phosphorylation, change in gene expression

and stimulation of cell proliferation.


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Another target for protein kinases is the transcription factor NF-

kB, which is involved in a variety of response of various aspects of

immune response .

The activity of NF-kB is regulated by translocation from cytoplasm

to nucleus. In inactive sate NF-kB is retained in cytoplasm as a

complex with an inhibitory subunit called IkB.

Activation of proetin Kinase leads indirectly to Phosphorylation of

Ikb which targets Ikb for proteolytic degradation.

Distruction of IKB allows NF-kB to translocate to nucleus where itregulates transcription of its target gene.


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Whereas diacaylglycerol remains attached to Plasma membrane, IP3

is a small polar molecule that is released into cytosol where it acts to

signal the release of Ca2+ from intracellular stores.

In cells ca2+ concentrator is kept low by Ca2+ pumps that export Ca2+

from cells and into ER ER serves as intracellular Ca+ store. IP3 binds

to receptor that are ligand gated ion channels. As a a result cytosolicCa2+ level increase to about 1 micro meter which affects the activity

of several target proteins.

Many effects of Ca2+ are mediated by Ca2+ binding Camodulin which

is activated at Ca2+ concentration upto 0.5 M. Ca2+/ Calmodulin

then binds to a variety of target proteins including Protein Kinases.


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One example of such Ca/Calmodulin dependant protein kinase is

myosin light chain kinase, which signals actin myosin contraction

by phosphorylation of Myosin light chain.

Other such Protein Kinase include member of CAM kinase IInd

Family, which phosphorylate a numberof protein including

metabolizing enzyme, ion channels and Transcription factors

One form of CAM kinase Iind is particularly abundant in nervous

system. It regulates synthesis and release of neurotransmitters.


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CAM Kinase II can regulate phosphorylating transcription factor.

One of transcription factor is CREB which is phosphorylated at

the same site by protein kinase A

In addition to cleavage by phospholipase C, PIP2 can be

phosphorylated on 3 position of Inositol by enzyme phosphatidyl

inositide (PI) 3 kinase. Like Phospholipase C one PI3 Kinase is

activated by G proteins, while second has SH2 domain and is

activated by receptor protein tyrosin kinase..


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Ras, Raf and MAP Kinase pathways

MAP Kinases are family of protein serine/ Threonine Kinase

called so far Mitogen activated protein kinases

In yeast, MAP Kinase pathways control cativity of cellular

responses including mating , cell shape and sporulation.

In higher eukaryotes, MAP Kinases are ubiqutous regulators of

cell growth and differentiation

Best characterize form of MAP Kinase in mammalian cells belongto ERK that act through either G protein Kinases family that act

through either g Protein receptor or protein tyrosin kinase.


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cell signalling lecture

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