Section 2: The Phosphoinositide Pathway

What are the products of phosphoinositide pathway

Produces 3 2nd messengers

1. Inositol-1,4,5-triphosphate (IP3)

2. Ca²⁺

3. 1,2-diacylglycerol (DAG)

Phosphoinositide pathway steps

1. Ligand binds to receptor

2. this activates heterotrimeric protein

   • GTP binds to ∂ subunit

3. G_q∂-GTP diffuses laterally along the plasma membrane

4. G_q∂-GTP activates the membrane-bound enzyme phospholipase C (PLC)

5. The actived PLC catalyzes the hydrolysis of phosphatidylinositol-4,5-biphosphate (PIP₂) at its glycero-phospho bond

6. PLC mediated PIP₂-hydrolysis yields inositol-1,4,5-triphosphate (IP₃) and 1,2-diacylglycerol (DAG)

PIP₂- Hydrolysis

• The hydrolysis of PIP₂ sets in motion both cytoplasmic and membrane-bound events

  • Diacylglycerol (DAG) acts as a 2^nd messenger in the membrane

  • IP₃ (charged, water-soluble) diffuses through the cytoplasm to the endoplasmic reticulum

  • IP₃ binds to and induces the opening of a Ca²⁺ transport channel in ER

    • efflux of Ca²⁺ from the ER

IP₃ induced Ca²⁺ release

• Efflux (the flowing out of a particular particle) of Ca²⁺ from the ER

  • Cytosolic [Ca²⁺] increase from ~0.1 µM to 10mM

• Increases [Ca²⁺]

  • this triggers glucose mobilization and muscle contraction via Ca²⁺-bondong protein calmodulin and its homologs

Calmodulin - a Ca²⁺ - Activated Switch

• Calmodulin (CaM): a small protein that detects calcium levels and sends signals to regulate many cellular functions

  • ubiquitous, eukaryotic Ca²⁺-binding protein

    • Ca²⁺-binding protein facilitates the binding of Ca²⁺ and can be present by itself

  • This participates in numerous cellular regulatory processes

Calmodulin (CaM) can function as

1. Free-floating monomeric protein

2. A subunit of a larger protein

• It is highly conserved 148-residue protein

• It is important to have the capability of binding to make it available to other proteins

CaM and globular domains

CaM has two structurally similar globular domains

• it is connected by a 7-turn ∂ helix

• Each globular domain contains 2 high-affinity Ca²⁺ - binding sites

  • in total 4 Ca²⁺

How are Ca²⁺-binding site formed

They are formed by nearly superimposable helix-loop-helix motifs known as EF hands

What do EF hands form

Ca²⁺-binding site in numerous other Ca²⁺-binding proteins

Ca²⁺-CaM mediated activation

(How is it activated?)

• Ca²⁺ binds to either domain of CaM

  • This induces a conformational change in that domain

    • When there is a ligand binding and can lead to a functional change

  • Exposed patch binds w/ high affinity to CaM-binding domains of Ca²⁺ -regulated protein kinases

What activates protein kinase C

DAG

Diacylglycerol (DAG)

• It is a lipid-soluble 2^nd messenger

• it remains embedded in the plasma membrane

• It actives the membrane protein kinase C (PKC)

• Increases the membrane affinity of PKC

• stabilizes PCK’s active conformation

What does an active PCK do

It phosphorylates several different cellular proteins

PKC (resting state)

phosphorylated in the cytosolic protein

What does both PKA and PKC phosphorylate

Ser & Thr residues

What destroys the 2^nd messengers

inositol polyphosphate 5-phosphatase

What proteins are associated directly by insulin

1. Shc

2. Gab-1

3. APS / Cbl

4. IRS proteins

After insulin binds, what do Shc and Gab-1 end up in

End up in DNA/RNA/protein synthesis

• This is for cellular growth and differentiation

Which is the protein has the most diverse route?

IRS

• They end up in

  • DNA/RNA/protein synthesis

  • Glycogen synthesis

  • Glucose transport

Insulin binding

• Insulin receptor autophosphorylates itself at several Tyr residues

• Then it Tyr-phosphorylates its target proteins

• Thereby activating several signaling pathways

IR-P- controls a diverse array of effects

1. Phosphorylation

   • Shc-P generates a binding site for Grb2’s SH2 domain

   • this causes a stimulation of a MAP kinase cascade

   • this ultimately affects growth and differentiation

2. Phosphorylation of Gab-1

   • This activates the MAP kinase cascade

• 1 & 2 end up in DNA/RNA/protein synthesis

Phosphorylation of insulin receptor substrate (IRS) proteins

• This activates enzymes known as phosphoinositide 3-kinases (PI3Ks)

• PI3K adds a phosphoryl group to to the ‘3’-OH group of a phosphatidylinositol

• 3-phosphorylated lipid activates protein kinase-1 (PDK1)\

An activated PDK1 initiates cascades leads to

• Glycogen synthesis

• Enhanced glucose transport

• Cell growth & differentiation

Phosphorylation of the APS / Cbl complex

• This stimulates TC10 (a monomeric G protein) and (PI3K-independent)

• regulation of glucose transport involving the participation of lipid rafts

How can a hormone, such as insulin trigger a variety of physiological effects

By activating multiple pathways

• This is not possible in a one-hormone one target regulatory system