Professor of Biochemistry

CELL SIGNALING

Dr.Saidunnisa M.D

Dr.Saidunnisa M.D

Learning Objectives At the end of the session student shall be

able to Define, explain and interpret :1. Hormone ,target tissue, receptor.2. Steroid hormone mechanism and clinical

interpretation.3. Zinc fingers.4. G-proteins, cAMP, mechanism of action,5. INP system as second messenger6. cGMP.

ENDOCRINE SYSTEMENDOCRINE SYSTEM The endocrine system is made up of glands called

ENDOCRINE GLANDS.it has a rich supply of blood with a relatively large number of blood vessels

A. THE HYPOTHALAMUS

B. PITUITARY GLAND

C. PARATHYROID

D. THYROID GLANDS

E. ADRENAL GLAND

F. PANCREAS

G. GONADS

secretes chemicals called hormones.

a ductless gland i.e. the hormone is secreted directly into the bloodstream.

Definition In Greek means to excite or to arouse. These are chemical substances produced by

the endocrine glands secreted into the blood stream carried to the target tissue where they act.

A small soluble organic molecule.

Travels in blood.

Fits into precise target molecule.

Effective in low concentration.

The hormones fall into two general classes based on their solubility in water.

The water soluble hormones are the ACTH, FSH, LH, MSH,TSH, ADH, HCG, PTH,CRH, catecholamine's, and peptide hormones.

Receptors are found on the target cell, on the plasma membrane.

These types of receptors are coupled to various second messenger systems which mediate the action of the hormone in the target cell.

The lipid soluble hormones include thyroid, steroid hormones and Vitamin D3

Receptors reside in the nucleus (and sometimes in the cytoplasm glucocorticoid) of

the target cell.

Because these hormones can diffuse through the lipid bilayer of the plasma membrane, their receptors are located on the interior of the target cell.

Classification Polypeptides: short chain AA <100 (Insulin and

ADH)Glycoprotein's: > than 100AA (FSH and LH)Amines: Amino acid derived from tyrosine and

tryptophan (epinephrine, norepinephrine and melatonin)

Steroids: derived from cholesterol (corticosteroids, androgens, estrogens and progesterones)

Classification based on mechanism of action

Group Mechanism of action Examples

IA Bind to cell surface receptors with cAMP second messenger

ACTH,FSH,LH,MSH,TSH,ADH, HCG, PTH,CRH, glucagon ,calcitonin, catecholamine's

IB Bind to cell surface receptors with cGMP second messenger

ANF

IC Bind to cell surface receptors with calcium or PIP2 as second messenger

TRH, GnRH, Acetylcholine, CCK, Gastrin, Vasopressin, Oxytocin

ID Bind to cell surface receptors mediated thro tyrosine kinases

Insulin, somatomedin, EGF,FGF, PDGF,IGF NGF

IE Having cell surface receptors, but intracellular messenger unknown

IL, GH, PRL, Erythropoietin

IIBind to intracellular receptors

Gluco / Mineralocorticoids,Estrogens, progesterones, androgens, calcitriol, thyroxine

Mechanism of Action of (Group-II) Hormones

1. These hormones are lipophilic in nature and can easily pass across the plasma membrane by simple diffusion.

2. They are transported in blood bound to serum albumin or specific transport protein (SHBG, TBG)

3. They bind to intracellular receptors (steroid hormone/thyroid hormone super family) located either in cytoplasm or nucleus.

Mechanism of Action of Steroid HormonesThe binding of hormone

to the receptor induces conformational change in size and shape of the receptor called Activation.

The hormone-receptor complex bind to the specific sequences on DNA called HRE and alter the gene regulation by transcription and translation.

Structure of Steroid Hormone Receptor1. Made up of multiple subunits.

2. Heat Shock Proteins: stabilizes the receptor prior to hormone binding.

3. Binding subunit has two areas of biological activity:

DNA binding region Hormone binding region

DNA binding regionHas about 70 A.A residues rich in cysteine, Arginine,

Lysine.For every 2 residues of cysteine and histidine 2

molecules of zinc bind.When zinc binds it protrudes 2 finger like projections

which are specific sites for DNA binding.This region is same in all the different steroid

hormone receptors.

Hormone binding region Has about 250 A.A starts after DNA binding

region.Rich in hydrophobic A.A and made up of alpha

and beta strands.This region is different in different members of

steroid hormone receptor family.This gives specificity for the particular hormone

and this property is made used in clinical medicine.

Tamoxifen Is used in the treatment of breast cancer. Division of cancerous cells express on the

continued presence of the hormone estrogen. Tamoxifen competes with estrogen in binding to

the estrogen receptor. Tamoxifen-receptor complex is inactive in gene

regulation.

Consequently Tamoxifen administration after surgery or chemotherapy for this type of breast cancer slows or stops the growth of remaining cancerous cells, prolonging the life of the patient.

RU486 This is a steroid analog used in the early

termination of the pregnancy. It is antagonist to hormone progesterone which is

required for implantation of ovum in the uterus. RU486 binds to the progesterone receptor and

bocks the actions essential for implantation.

Proteins involved in Group-I Hormone Action1. Hormone receptor.

2. Gs/i proteins.

3. Adenylate cyclase.

4. cAMP.

5. Phosphodiesterase.

G-ProteinsAlfred G. Gilman and Martin Rodbell were

awarded Nobel prize for their work on G proteinsG stands for Guanyl nucleotide they are bound to

GDP/GTP.It is a peripheral protein.It is a trimer having three subunits α β .

G-Proteins The interaction of the hormone with its receptor

results in the activation or inactivation of adenylate cyclase.

This process is mediated by 2GTP dependent regulatory proteins Gs and Gi.

The α subunit of the Gs and Gi are different but β subunits are same.

G-Protein-Coupled Receptors (GPCRs)

These are trans membrane proteins that wind 7 times back and forth through the plasma membrane (heptahelical receptors).

Their ligand-binding site is exposed outside the surface of the cell.

In the inactive state Gα has GDP in its binding site.

How they work When an hormone binds to

the associated GPCR an allosteric change takes place in the receptor (in its tertiary structure)

This triggers an allosteric change in Gα causing:

GDP to leave and be replaced by GTP

GTP activates Gα causing it to dissociate from G β G.

How they work Activated Gα in turn

activates adenylyl cyclase bound to plasma membrane which activates conversion of ATP to cAMP (2nd messenger) resulting in amplification of hormonal cascade.

Phosphodiesterase

cAMP is degraded by Phosphodiesterase.

This is inhibited by caffeine and theophyllin.

Protein Kinases-A This is an hetero tetramer

made of 2 regulatory (R) and 2 (C) catalytic molecules at rest it is in inactive form.

cAMP binds to the inactive protein kinases and disassociate R subunits from C subunits.

The active subunit is C catalyses phosphorylation of proteins and ultimately causes the biochemical function.

Hormone action thro Protein Kinases-A

cAMP mediated glycogenolysis

Stimulatory and inhibitory pathways

Second Messenger as cGMPOne hormone uses this as second messenger

(ANP Atrial Natriuretic peptide or ANF Factor)

cGMP is formed from GTP by the enzyme Guanylate cyclase which is membrane bound.

cGMP is also involved in the rhodopsin cycle (vision, visual cycle)

cGMP In heart cGMP causes less forceful contractions

by stimulating ionic pump that maintain low cytosolic calcium concentration (relaxation) and vasodilatation.

Nitroglycerine and nitroprusside act by inhibiting Phosphodiesterase.

This is the same response brought by nitroglycerine tablets taken for angina and nitroprusside taken for BP.

Atrial natiuretic peptide Atrial natiuretic peptide (ANP),this hormone is

secreted by cardiac muscle cells.

Released by muscle cells in the atria in response to high blood pressure.

Involved in homeostatic control of body water, sodium, and potassium.

ANP might act on tubule cells to increase sodium excretion.

Natriuresis refers to enhanced urinary excretion of sodium.

Phosphotidyl Inositol as Second Messenger

PI4,5bisphos is cleaved by PLC to generate the two intracellular messengers,

DAG and IP3.PI3,4,5 serves as docking

site for PH domain.

Phosphotidyl Inositol as Second Messenger

INP3 passes through the cytoplasm and stimulates the ER to release Calcium.

DAG is nonpolar activates membrane bound protein kinase C for which calcium is required.

Synergism between INP3 and DG is to phosphorylate the target proteins through Protein kinase C and Calmodulin.

Calmodulin Therefore some consider Calmodulin as

second messenger in PI system.Calcium in turn through calmodulin

contributes to a variety of biochemical functions like:

Smooth muscle contraction, Glycogen breakdown, and Exocytosis.INP3 has short half life.

Stimulatory pathway

Cholera A toxin liberated by vibrio cholera binds to G αs subunit of heterotrimeric G-proteins.

Thereby keeps it “turned on”.

The resulting continuous high levels of cAMP activates the CFTR channel, resulting in secretion of chloride ion and Na+ ion into the intestinal lumen.

The ion secretion is followed by loss of water, resulting in vomiting and watery diarrhea.

CFTR-gated channel Cholera toxin binds to the

Glycocalyx of gycolipid called GMI-Ganglioside of intestinal mucosal cells.

Cholera toxin absorbed into the intestinal mucosal cells, it is processed and complexed with Arf (ADP-ribosylation factor) activates Phosphorylation of the regulatory domain of CFTR

by protein kinase A.

Stimulatory pathway Glucagon and Glucocorticoids: stimulate

gluconeogenesis

Epinephrine and glucagon: stimulate Glycogenolysis.

Inhibitory pathway

B. pertussis is a Gram-negative, aerobic coccobacillus responsible for the disease whooping cough. Whooping cough, a highly contagious disease

Pertussis toxin binds to G αi subunit of G-proteins, This inhibits the adenylate cyclase and causes low

levels of cAMP.

Thereby keeps it “turned off”. Glucocorticoids: inhibit glycolysis

Kinase receptorsThree types:

1.Tyrosine kinase receptors

2.JAK-STAT receptors (janus kinase/ singnal transducer and activator of transcription).

3.Serine/threonine kinase receptors.

Tyrosine Kinase receptorsInsulin receptor exists

as a preformed dimer.Each half containing

alpha and beta subunits.Autophosphorylate each

other when insulin binds and thereby activates the receptor.

Activated phosphorylated receptor binds to the IRS (insulin receptor substrate)

Insulin actionIRS is Phosphorylated

at multiple sites creating multiple binding sites for proteins Grb2, phospolipase C (PLC ) and PI-3kinase.

Insulin action PI-3 binds and phosphorylates PI-4,5 bis phoaphate to Pi-3,4,5P

Protein kinase B and PDK1 (phosphoinositide dependent protein kianse) are recruited to the membrane where PDK1 phosphorylates and activates protein kinase B

Case 25 year old woman suffers from anorexia nervosa,

has increased her weight on the advice of her physician, she has been eating more to prevent fatigue during her daily jogging regimen.

She runs about 10miles before her breakfast every second day and forces herself to drink high –energy supplement immediately.

List the hormones taking part in the above patient?Name the mechanism of action of the above

mentioned hormones?Name the Metabolic pathways involved

AnswerOvernight fast alpha cells of pancreas secretes

glucagon.Stress of prolonged fasting and jogging

stimulates the secretion of :Cortisol, epinephrine and nor epinephrineAll the above hormones act on different target

tissues by binding to different receptors and work differently .

Cortisol acts by binding to the intracellular receptors increase transcription of the genes for GNG.

Glucagon binds to heptahelical receptors in liver and adipose tissue act through cAMP and protein kinase A.

Insulin released when she drinks high energy supplement works through tyrosine Kinase receptor.

Epinephrine and nor epinephrine released during her exercises act by binding to heptahelical receptors in liver and adipose tissue act through cAMP and protein kinase A.

MECHANISM OF HORMONE ACTIONMECHANISM OF HORMONE ACTION

i. Gene activation ii. Second messenger (cAMP)

Steroid hormone

Non- steroid hormone

COMPARISON BETWEEN THE TWO MECHANISMS OF HORMONAL ACTION

Cyclic AMP (cAMP) Gene activation

Most of the non-steroid hormones such as peptide hormones reacted by cAMP

Steroid hormones activate gene to produce effect

Short term effect (ranging from a few minutes to a few hours)

Long term effect (ranging from a few hours to a few days)

COMPARISON BETWEEN THE TWO MECHANISMS OF HORMONAL ACTION

Cyclic AMP (cAMP) Gene activation

Hormones bind to the protein receptors that are burrowed on the targeted cell membrane

Hormones bind to protein receptors that are inside the nucleus or cytoplasm

Hormones are stationed outside the cells and are unable to enter the cells

Hormones pass through the plasma membrane and enter into the target cells

COMPARISON BETWEEN THE TWO MECHANISMS OF HORMONAL ACTION

Cyclic AMP (cAMP) Gene activation

Hormones do not activate the gene

Hormones that are bonded to receptors activate gene

No transcription and translation take place

Transcription and translation gene are activated

Protein kinases inside the cell are activated by cAMP

Protein kinases are not activate

Cascade effect amplify the hormone’s affect

Cascade effect does not form