HormonesG Protein’s 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 02

Time : 10:00am

Dated: 07/03/2015

The 2012 Nobel Prize in Chemistry was awarded jointly to

Robert J. Lefkowitz (l) and Brian K. Kobilka

for

studies of G-protein-coupled receptors

G Protein Coupled Receptor’s (GPCR’s)

• They are the receptor named just because they interact with G Protein’s.

• Also referred to as “Serpentine Receptor’s” as they contain seven trans-membrane helices (7TM)

• Largest known receptor family – Constitutes > 1% of the human genome.

• Comprises receptors for a diverse array of molecules: neurotransmitters, odorants, lipids, neuropeptides, large glycoprotein hormones

G Protein Coupled Receptor’s (GPCR’s)

• Their Structure consists of:

Amino- Terminal: Present on the outside of the cell

Seven α helices: traversing the plasma membrane & connected by loops at varying length

Carboxyl-Terminal: Present on Inside of the cell

• Ligand binding site: three loops that are on outer surface of the cell

• Docking site: three loops that are on inner surface/ cytoplasmic side of the cell; provide site for binding of intracellular protein – G protein’s

G Protein Coupled Receptor’s (GPCR’s)

G Protein Coupled Receptor’s (GPCR’s)

• They are named so because they bind to guanine nucleotide as prosthetic group.

GDP – Inactive form

GTP – Active form

• Hetero-trimeric in nature : Three different polypeptide subunits – α, β & γ.

• Held at plasma membrane by lipid chains that are covalently attached to the α & γ.

G Protein

• The guanine nucleotide binding site is present on Gαsubunit:

In GDP bound conformation (Inactive, b): Gα subunit has high affinity for the Gβγ; hence they remain together as trimer on cell surface

In GTP bound conformation (Active, a): : Gα subunit has low affinity for the Gβγ; leading to its dissociation from complex

• The two conformations are inter-convertible via activation by GPCR’s; which cause GDP-GTP switching on Gα subunit of trimer.

• Each dissociated Gα subunit in turn is free to activate an effector protein – Like “Adenylyl Cyclase” etc.

G Protein

G Protein

• Thus, Gα subunit is said to be “On” when it is bound to GTP.

• Gα subunit turn itself “Off” by hydrolysis of bound GTP via its intrinsic GTPase activity.

• The Gα subunit is categorized into four types: Gs: Stimulatory: ↑cAMP levels; ↑Adenylyl cyclase activity; ↑ Cardiac Ca2+

Gi: Inhibitory: ↓ cAMP levels ; ↓Adenylyl cyclase activity; ↑ K+ Channels

Gq: Stimulatory: ↑Phopholipase C- B1; ↑IP3; PIK3

G12/13: Unknown: ↑Cl- Channels

• GEF: Guanosine Exchange factors: cause GTP – GDP switching

• GAP: GTPase activating Protein: Increases the rate of GTP hydrolysis

• RGS: Regulators of G protein Signaling: Increases the rate of GTP hydrolysis

G Protein

G Protein

The G Protein Cycle

The G Protein Cascade

• Cyclic AMP

• Cyclic GMP

• Inositol Tri Phosphate - IP3

• Diacyl glycerol - DAG

• Ca/Calmodulin

• Smad

Second Messengers

• Are small easily diffusible chemicals/intermediates

• Have very short half life

• They relay signals received at the receptors on the cell surface

• Serve to enhance the strength of the signal

• Affect more than one pathway and/ or protein

• Cause divergence of the signal

Second Messengers

Cyclic AMP Cyclic GMP Ca2+ Diacylgycerol Protein

substrates

PK-A PK-G Calmodulin PK-CProtein Ser/Thr

kinases

Protein substrates

Protein substrates

Protein substratesMultifunctional

kinasesOther

phospholipases

1 2 3 4 5

1 2 3 4 5

Tyrosine

kinase

IP3G G G G

InsulinGlucagonT-cell

Activation

Nitric

oxide

G protein

End result is

phosphorylation of

one or more proteins

Benefits of a 2° messenger system

Amplification

Signal molecule Receptor protein Activated adenylyl cyclase

Amplification

Amplification

Amplification

Amplification

GTP G protein

2

1

3

4

5

6

7

Enzymatic product

Enzyme

Protein kinase

cAMP

Not yetactivated

• Produced by activation of Adenylyl Cyclase by Gα from ATP.

• Has capability to easily diffuse to other sites within the cell.

• cAMP molecules diffuse into cytoplasm and bind to regulatory subunits of cAMP dependent “Protein Kinase A”

• Protein Kinase A is its effector protein via which cAMPmediates its function

cAMP

Cyclic AMP System

Receptor

Adenylate cyclase

G-protein

Protein Kinase A

c-AMP

Stimulate (Gs) and

Inhibit (Gi)

cAMP

cAMP is generated from ATP by an enzyme: adenylyl cyclase. AC is regulated by G proteins

cAMP

cAMP activates one or more kinases.

• Is a hetero-tetramer –• Two regulatory & two catalytic subunits (R2C2).

• The regulatory subunits normally inhibit the catalytic activity of enzyme.

• The cAMP binding to regulatory subunits causedissociation of regulatory subunits from the tetramer

• Therefore, results in release of the catalytic subunits of PKA for further downstream function.

Protein Kinase A - PKA

• PKA either function to regulate the metabolism by phosphorylating the key metabolic enzymes like: Glycogen Phosphorylase, Gylcogen Synthase etc.

Activates Glycogen PhosphorylaseUsed in utilisation of glycogen, degrades it to release glucose

in blood stream

Inactivates Glycogen SynthaseUsed in assimilation of glucose to glycogen, uptakes glucose

from blood stream and stores in liver & muscles as glycogen

Activates Phosphorylase Kinase

Protein Kinase A - PKA

• Or some of it translocates to the nucleus

• In nucleus it phosphorylates key nuclear proteins which function as transcription factor called CREB (cAMP Response element Binding- Protein)

• CREB forms dimer and then binds to the DNA at particular sequences within the promoter/regulatory region 5-TGACGTCA known as CRE (cAMP Response Element)

Protein Kinase A - PKA

Protein Kinase A & Its Effects

Cholera toxin catalyzes covalent modification of Gs.

• ADP-ribose is transferred from NAD+ to an arginine residue at the GTPase active site of Gs.

• ADP-ribosylation prevents GTP hydrolysis by Gs.

• The stimulatory G-protein is permanently activated.

Pertussis toxin (whooping cough disease) catalyzes ADP-ribosylation at a cysteine residue of the inhibitory Gi, making it incapable of exchanging GDP for GTP.

• The inhibitory pathway is blocked.

ADP-ribosylation is a general mechanism by which activity of many proteins is regulated, in eukaryotes (including mammals) as well as in prokaryotes.

Toxic Effects

Questions