Biochemical signaling

SOME FEATURES OF SIGNAL

TRANSDUCTION
 SENSITIVITY

Receptor has high affinity (Low Km) for signaling ligand (L). Its means

that the receptor detects micromolar to nanomolar concentration of a

signaling ligand.
AMPLIFICATION
Modularity (separated or recombined)

Protein with multivalent

affinities form diverse
signaling complexes from
interchangeable parts.
Phosphorylation provide
reversible points of
interaction.
Four General types of Signal Transducer
 G- Protein Coupled Receptors
G-protein coupled receptors (GPCR) that acts through a member of
guanosine nucleotide – binding protein or G- protein.

Three essential component for signal transduction thru GPCR are..

1. Plasma membrane Receptor (Seven pass receptor)

2. G-protein that cycles between active and inactive state.

3. An affecter enzyme or ion channel.

 G- Protein Coupled Receptors
 Human genome encodes 800 GPCRs.

 out of which 350 detect hormones, growth factors.

 Upto 500 hundred are serves as gustatory (taste) and olfactory (smell)
receptors.

 GPCR receptors are implicated in many disease condition like diabetes,

cardiovascular defects.

 β-adrenergic receptor mediate epinephrine, target of most β-blockers.

G- Protein Coupled Receptors
 A (GPCR) Β-adrenergic receptor a prototype
 Epinephrine release from adrenal gland give fight, flee response thru GPCR.

 Four Types of adrenergic receptor ( α1, α2 , β1, β2 ).

 Different response to agonist and antagonist against these receptor.

 what are Agonist ?...Natural ligand or Structural analog which initiate response

 what are antagonist?...An analog which does not initiate response.

 A (GPCR) Β-adrenergic receptor a prototype
 β1, β2 adrenergic receptor are present on liver, muscle and adipose tissue.

 These are involve in fuel metabolism of fat and glycogen.

 GPCR receptors are integral protein, 7 helical regions of 20 to 28 A.A.

 These receptors passes seven times thru the membrane.

 G- protein are heterotrimeric having α, β, γ subunits.

 GPCR are allosteric protein.

 A (GPCR) Β-adrenergic receptor a prototype
 Allosteric protein when bind to ligand undergo transition.

 This transition favour the replacement of GDP with GTP.

 G protein in GTP bound form become active and called stimulatory (Gs)
protein.

 Now β and γ subunits of Gs dissociate from the α subunit as βγ dimer and Gsα

 with its bound GTP. This Gsα move in the plane of membrane and activate
Adenylyl cyclase nearby .
 A (GPCR) Β-adrenergic receptor a prototype
 Adenylyl Cyclase (AC) is an intergral membrane protein, on association of Gsα

 it become activated and converts ATP to cAMP hence raising level of cAMP.

 Stimulation of Gsα is self limiting. It has its own GTPase activity and convert
GTP to GDP thus rendering the activated AC to inactive state.

 Gsa will unbound from AC and reassociate with βγ dimer and inactive Gs will
be again available to interact with hormone bound receptor.
 A (GPCR) cAMP activates PKA

 Increased cAMP level will in turn activate Protein kinase A (PKA), an

enzyme which results in phosphorylation of many enzymes at serine and

threonine residue like glycogen phosphorylase b kinase which results in

mobilizing the glycogen stores in muscle and liver in response to epinephrine

for energy need.

 Structure of PKA
 Protein kinase A (PKA) has two identical subunits.

1. Regulatory subunit (R)

2. Catalytic subunit (C)

 During inactivation state these both subunits join with each other in such a way
that catalytic region on catalytic subunit is inhibited by regulatory subunit. But
when cAMP level rises and cAMP binds with the regulatory subunits pulling its
inhibitory sequence out of catalytic subunit rendering it free, as active catalytic
subunit.
Structure of PKA
MECHANISM OF GPCR
 TERMINATION OF GPCR RESPONSE
 There are four ways in which signal termination occur..

1. The response to epinephrine stimulation will end when the conc of ligand in
the blood drops below the Kd (distribution coefficient) for its receptor and the
later reassume its inactive conformation in which it can no longer activate Gs.

2. A second means of ending the response is the hydrolysis of GTP bound to the
Gα subunits catalysed by the GTPase activity of the G protein. Conversion of
bound GTP to GDP Favors the returns of Ga to conformation which it bind to
Gβγ subunits.
 TERMINATION OF GPCR RESPONSE
3. Thirdly, the metabolic effects that results from phosphorylation of target enzyme by
PKA are reversed by the action of phosphatases which hydrolyzed phosphorylated Tyr,
Ser or Thr residue releasing inorganic phosphate.

4. Above termination signals take effects when signal ends. A different desensitization
method is also used when even signal persist.

i. When the receptor remains occupied with epinephrine, βadrenergic receptor

kinase, or βARK, is drawn to the plasma membrane by its association with the Gs βγ
subunits and is thus positioned to phosphorylate the receptor.
 TERMINATION OF GPCR RESPONSE
ii. Receptor phosphorylation creates a binding site for the protein β-arrestin, or
βarr. , and binding of βarrestin blocks the sites in the receptor that interact with
the G protein.

iii. The binding of β-arrestin also facilitates the sequestration of receptor

molecules and their removal from the plasma membrane by endocytosis into
small intracellular vesicles (endosomes).
 TERMINATION OF GPCR RESPONSE
iv. The arrestin-receptor complex recruits clathrin( a contractile protein) and
other proteins involved in vesicle formation, which initiate membrane
invagination, leading to the formation of endosomes containing the adrenergic
receptor.

v. In this state receptor become inactive, but after dephosphorylation of

receptor, it returned to the plasma membrane and resensitizing the system to
epinephrine.
TERMINATION OF GPCR RESPONSE
 cAMP act as Second Messenger
 Glucagon binds to its receptors in the plasma membrane of adipocytes, activating (via a

Gs protein) adenylyl cyclase. PKA, stimulated by the resulting rise in [cAMP],

phosphorylates and activates two proteins critical to the mobilization of the fatty acids of

stored fats.

 Some hormones act by inhibiting adenylyl cyclase, thus lowering [cAMP]. The binding of

somatostatin to its receptor in the pancreas leads to activation of an inhibitory G protein,

or Gi , that inhibits adenylyl cyclase and lowers [cAMP]. In this way, somatostatin inhibits
 cAMP act as Second Messenger

 In adipose tissue, prostaglandin E2 inhibits adenylyl cyclase, thus lowering

[cAMP] and slowing the mobilization of lipid reserves triggered by epinephrine

and glucagon.

 In certain other tissues, PGE2 stimulates cAMP synthesis: its receptors are

coupled to adenylyl cyclase through a stimulatory G protein, Gs. In tissues with

α2-adrenergic receptors, epinephrine lowers [cAMP].

 Adaptor Proteins

 Another factor that explains how so many types of signals can be mediated

by a single second messenger (cAMP) is the confinement of the signaling

process to a specific region of the cell by adaptor proteins.

Adaptor Proteins
 DAG, IP3 and Ca+2+ Role as 2nd Messenger

 A second broad class of GPCRs is coupled through a G protein to a

plasma membrane phospholipase C (PLC) that catalyzes cleavage of

the membrane phospholipid phosphatidylinositol 4,5- bisphosphate,

or PIP2 . Different Steps involve are as under…

1. The receptor-hormone complex catalyzes GTP-GDP exchange on an

associated trimeric G protein, Gq.

 DAG, IP3 and Ca+2+ Role as 2nd Messenger

2. The activated Gq activates the PIP2-specific PLC.

3. It catalyzes the production of two potent second messengers

diacylglycerol (DAG) and inositol 1,4,5-trisphosphate, or IP3.

4. IP3, diffuses from the plasma membrane to the endoplasmic

reticulum (ER), where it binds to the IP3-gated Ca2+ channel, causing

the channel to open Ca2+ rushes into the cytosol.

 DAG, IP3 and Ca+2 Role as 2nd Messenger

5. Diacylglycerol cooperates with Ca2+ in activating PKC, thus both acting

as a second messenger.

6. The ultimate PKC targets include cytoskeletal proteins, enzymes, and

nuclear proteins that regulate gene expression.

7. Compounds that lead to overexpression of PKC or that increase its

activity to abnormal levels act as tumor promoters.

DAG, IP3 and Ca+2 Role as 2nd Messenger
 GPCR in Vision, Olfaction, and Gustation

Vision, olfaction, and gustation in vertebrates employ GPCRs, which

act through heterotrimeric G proteins to change the membrane

potential (Vm) of a sensory neuron.

 GPCR in Vision, Olfaction, and Gustation

 Light activates the GPCR rhodopsin, which allosterically activates

the trimeric G protein transducin. Tα activates a cGMP

phosphodiesterase, lowering [cGMP] and closing cGMP dependent

ion channels(Na and Ca ions). The membrane become hyperpolarized

resulting electrical impulse carries the signal to the brain.

 GPCR in Vision, Olfaction, and Gustation

 In olfactory neurons, olfactory stimuli, acting through GPCRs and G

proteins, trigger an increase in [cAMP] (by activating adenylyl cyclase)

or [Ca+2] (by activating PLC). These second messengers affect ion

channels and thus the Vm. Gustatory neurons have GPCRs that respond

to tastants by increasing levels of cAMP, which changes membrane

potential by closing K+ ion channels.

Common features of signaling system
 Receptor Tyrosine Kinase

 The receptor tyrosine kinases (RTKs), a family of plasma membrane receptors with

protein kinase activity, transduce extracellular signals by a mechanism fundamentally

different from that of GPCRs.

 The active insulin receptor protein (INSR) consists of two identical α subunits

protruding from the outer face of the plasma membrane and two transmembrane β

subunits with their carboxyl termini protruding into the cytosol — a dimer of αβ

monomers
Receptor Tyrosine Kinase (Insulin Receptor)
 Receptor Tyrosine Kinase (Insulin Receptor)

• The α subunits contain the insulin-binding domain, and the intracellular

domains of the β subunits contain the protein kinase activity.

• Signaling through INSR begins with the binding of one insulin molecule

between the two subunits of the receptor on the extracellular side, causing

movement of the Tyr kinase domains together and stimulating their activity
 Receptor Tyrosine Kinase (Insulin Receptor)

• Each β subunit phosphorylates three essential Tyr residues near the carboxyl

terminus of the other β subunit. This autophosphorylation opens the active

site so that the enzyme can phosphorylate Tyr residues of other target

proteins.
 Steps involve in Signal Transduction

Different steps involve in insulin signal transduction are as under.

1. Insulin receptor bind insulin and undergo autophosphorylation on its

carboxy terminal Tyr residue.

2. Insulin receptor phosphorylate IRS1 on its residue.

3. SH domain of Grb2 binds to P- Tyr of IRS1. SOS binds to Grb2 then to Ras
causing GDP release and GTP binding to Ras.

4. Activated Ras binds and activates Raf-1.

5. Raf 1 phosphorylate MEK on two Ser residue activating it.

 Steps involve in Signal Transduction

6. MEK phosphorylate ERK on the Thr and Tyr residue activating it.

7. ERK moves into the nucleus and phosphorylate nuclear transcription factors
such as ElK1 activating them.

8. Phosphorylated ElK1 joins SRF to stimulate the transcription and translation

of a set of genes needed for cell division.

 Raf1, MEK and ERK belongs to family of larger kinases MAPK (mitogen
activated protein kinase). Raf1 belong to MAPKKK, MEK to MAPKK and ERK to
MAPK.
 Steps involve in Signal Transduction

 MAPK cascade (Such as Raf-MEK-ERK) mediate signaling initiated by a variety

of growth factors PDGF and EGF.
Steps involve in Signal Transduction
 CROSS TALK AMONG SIGNALING SYSTEM
 There is extensive cross talk among signaling systems.

 The regulatory circuitry that governs metabolism is richly interwoven and

multilayered.

 Insulin opposes the metabolic effects of epinephrine in most tissues, and

activation of the insulin signaling pathway directly reduces signaling through

the β-adrenergic signaling system.

 CROSS TALK AMONG SIGNALING SYSTEM

 When INSR is activated by insulin binding, its Tyr kinase directly phosphorylates

the β-adrenergic receptor (right side) on two Tyr residues (Tyr 350 and Tyr364) near

its carboxyl terminus, and indirectly causes phosphorylation of two Ser residues

in the same region (through activation of protein kinase B (PKB). The effect of

these phosphorylations is internalization of the adrenergic receptor, reducing the

response to the adrenergic stimulus.

 CROSS TALK AMONG SIGNALING SYSTEM

 Alternatively (left side) INSR catalyses phosphorylation of a GPCR on a carboxy

terminal Tyr creates the point of nucleation for activating the MAPK cascade

with Grb2 serving as the adaptor protein. In this case INSR use GPCR receptor

to enhance its own signaling.

 CROSS TALK AMONG SIGNALING SYSTEM