Section 2: Heterotrimeric G proteins

Heterotrimeric G proteins

• They are members of the superfamily of regulatory GTPases that are collectively known as G proteins

• G proteins share a common structural motifs

• They bond to guanine nucleotides GTP and GDP

• Hydrolyze GTP → GDP + P_i

Why are heterotrimeric G proteins essential

• Signal transduction

  • conveying a signal from exterior to interior of the call and / or cell to target

• Vesicle trafficking

• The growth of actin mircofilaments

• Translation

• protein targeting

  • as components of singal recognition particle (SRP) and the SRP receptor

Many heterotrimeric G proteins participate in signal transduction that consist of 3 major components

1. G protein-couples receptors (GPCRs)

2. Heterotrimeric G protein

3. Adenylated cyclase (AC)

G protein-coupled receptors (GPCRs)

They are transmembrane proteins that bind their corresponding ligand on their extracellular side and induces a conformational change on their cytoplasmic side

Where are heterotrimeric G proteins anchored to

Anchored to the cytoplasmic side of the plasma membrane

How are heterotrimeric G proteins activated

They are activated by a G-protein-coupled receptor (GPCR) when it binds to its corresponding ligand

• This receptor recognizes / detects a signal from the exterior of the cell ams transfers via autophosphorylation to the interior of the cell and there is the G-protein

Where do heterotrimeric G-proteins convey its information to

Adenylate cyclase (AC)

Adenylate cyclase

• It is bound to the transmembrane enzyme

• It can be activated or inhibited by activated heterotrimeric G proteins

What does activated adenylate cyclase catalyze

It catalyzes the synthesis of adenosine-3’,5’-cyclic monophosphate (cAMP) from ATP

• it facilitates the release of 2 phosphates

• converts ATP → cAMP

ATP molecule

cAMP

• cyclic adenosine monophosphate (cAMP)

• it binds to a variety of proteins and activates numerous cellular processes

• They act as secondary messengers

• they are polar, freely

Adenylate cyclase active signaling system

1. Receptor protein recognizes external signal and is bound to the inactive heterotrimeric G-protein

2. ∂-unit exchanges GDP w/ GTP and it is now active

3. ∂ will dissassociate from ß and γ subunits and at the same time, it moves towards the adenylate cyclase

4. The activity conveyed by AC is the conversion of ATP → cAMP

Adenylate cyclase inhibitng signaling system

1. Inactive form binds to the receptor

2. GDP is replaced w/ GTP

3. ∂-subunit has an inhibitory effect

How many isoforms of adenylated do mammals have

9

What are AC isoforms

tissues specific and have different regulatory properties

AC are transmembrane glycoprotein s

What are the conserved domains of Adenylated cyclase

1. Small N-terminal domain (N)

2. Transmembrane domane (M₁)

3. 2 consecutive cytoplasmic domains (C_1a & C_1b)

4. Transmembrane domain (M₂)

5. 2 consecutive cytoplasmic domans (C_2a & C_2b)

C_1a & C_2a

• They form the catalytic core

• They are 40% identical which allows them to associate w/ each other

• C_1a, C_1b & C_2a bind regulatory molecules

• Cells can adjust their cAMP levels in response to a great variety of stimuli

  • depending on the concentration of cofactors, it allows the cell to regulate the amount of ATP we want to convert

What is the target for cAMP

Protein Kinase A (PKA)

Protein Kinase A (PKA)

• It is made up of 4 subunits

  • 2 regulatory subunits

  • 2 catalytic subunits

• PKA is inactive when all four are bound (PKA heterotetramer)

• cAMP binds to the regulatory subunits

  • this causes dissociation of active catalytic monomers

What happens when cAMP binds to the regulatory subunit

• It could result in the disassociation of the regulatory subunit from the catalytic subunit

• The catalytic subunit then facilitates the phosphorylation of the target protein

Protein Kinase A chemical formula

Intracellular concentration of cAMP

• it determines the fraction of PKA in its active form

• it is the rate at which it phosphorylates its substrates

Regulatory subunit of protein kinase A

• it competitively inhibits its catalytic subunit

  • When the regulatory subunit is bound to the catalytic subunit, the entire protein is inactive

• contains two homologous cAMP-binding domains (A & B)

• also contains autoinhibitor segment

  • thsi resembles the catalytic subunit substrate

• We can inhibit catalytic subunit by the absence of cAMP

  • once we have cAMP, they can start to bind w/ A & B domains and can no longer bind to catalytic subunit, therefore will be released

Inactive R₂C₂ complex

• The authoinhibitor segments binds in the catalytic subunit’s active site

  • as does the inhibitory peptide → blocks substrate binding

Each R subunit cooperatively binds to

2 cAMP

What happens when the B domain lacks bound cAMP

• It masks the A domain

• It prevents A domain from binding cAMP

What happens when there is a binding of cAMP to the B domain

• It triggers a massive conformational change

• The A domain can bind cAMP

What happens if the A-domain is bound to cAMP

• The release of the now-active C subites from the complex

How does cAMP activates the PKA

• Activates by binding to the regulatory dimer

• When it binds, the catalytic subunit C dissociates

• This activates various cellular proteins by catalyzing their phosphorylation

• Singaling is limited by the action of phosphatases and cAMP phpsphodiesterase

What limits the second messenger activity

1. Phosphatases

2. cAMP phosphodiesterase

   • are chemically based signaling system

   • the signal molecule must eventually be eliminated

     • in order to control the amplitude and duration of the signal

     • and to prevent interference with the reception of subsequent signal

cAMP-phosphodiesterases

hydrolyzes cAMP to AMP

PDE superfamily

• included both cAMP-PDEs and cGMP-PDEs

• they are encoded by at least 20 different genes groups into 12 families