ABI 103 Section 2 Subsection 4 "The phosphoinositide Pathway"

The Phosphoinositide Pathway

• production of 3 2nd messengers that are involved in many pathways

the 3 secondary messengers produced are

• inositol-1,4,5-trisphophate (IP3)

• Ca²+

• 1,2-diacylglycerol (DAG)

Ligand binding results in

• the cytoplasmic release of the 2nd messenger Ca²+ and IP3

Ligand binds to receptor causing activation of

hetertrimeric G protein ( GTP binds to alpha subunit)

Gqalpha-GTP diffuses laterally along plasma membrane and activates…

• the membrane bound enzyme phospholipase C (PLC)

Activated PLC catalyzes the

• hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) at its glycerol-phospho bond

PLC mediated PIP2 hydrolysis yields

• (IP3)

• DAG

Hydrolysis of PIP2 sets in motion Both

• membrane bound and cytoplasmic events

Where does Diacylglycerol (DAG) act as a 2nd messenger ?

• in the membrane

Where does IP3 diffuse through?

• charged and water soluble

  • so through the cytoplasm → ER

How does IP3 associate with Ca²+?

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

  • efflux of Ca²+ from ER

Cytosolic [Ca²+] increases causing

• muscle contraction, actin filaments

• triggers glucose mobilization

Calmodulin (CaM) is a

Ca²+ activated switch

CaM

• highly conserves 148- residue protein

• ubiquitous

• eukaryotic Ca²+ binding protein (facilitates binding)

• in numerous cellular regulatory processes

CaM can function as

1) free floating monomeric protein alone

2) as a subunit of a larger protein domain

CaM has 2 structurally similar globular domains

• connected by seven-turn alpha helix

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

EF Hands CaM

• each Ca²+ binding site is formed by nearly superimposable helix-loop-helix motifs (EF Hands)

EF Hands form

• Ca²+ binding sites in numerous other Ca²+ binding proteins

Ca²+- cAM binding to either domain of CaM induces

• conformational change in that domain

• functional change can block/access active site

• exposed patch binds with high affinity to caM-binding domains of Ca²+-regulated protein kinases

DAG

• lipid-soluble 2nd messenger

DAG location

• embedded in plasma membrane

DAG activates

• membrane- bound protein kinase C (PKC)

Active PCK

phosphorylates several different cellular proteins

PKC resting state

• phosphorylated

• cytosolic protein

DAG increases membrane affinity of

• PKC

• stabilizes PKCs active conformation

BOTH PKA and PKC phosphorylate

• Ser and Thr residues

Phosphoinositide System is limited by

• destruction of its 2nd messengers

• ex: IP5 phosphotase

Inositol polyphosphate 5-phosphatse

IP3→ IP2

• removal of 2nd messenger from pathway

• disturbs the Ca²+ transport channel

Insulin Signaling Pathway

• once insulin gets phosphorylated all proteins get phosphorylates

Insulin Binding Step 1

• insulin receptor autophosphorylates itself at Tyr residues

• Tyr-phosphorylates its target proteins

  • activating several signaling pathways

IR-P controls diverse effects: DNA/RNA/Protein Synthesis

1) phosphorylation of the adaptor protein Shc

2) phosphorylation of Gab-1

phosphorylation of the adaptor protein Shc

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

• stimulation of a MAP kinase cascade

• affecting growth and differentiation

Phosphorylation of Gab-1

• activates the MAP kinase cascade

IR-P controls diverse effects: glycogen synthesis/ glucose transport/cell growth and differentiation

3. Phosphorylation of insulin receptor substrate IRS proteins

Phosphorylation of insulin receptor substrate IRS proteins

• activates enzymes known as PI3Ks

PI3Ks add

• phophoryl group to the ‘3’-OH group of phosphatidylinositol

• 3-phophorylated lipid activates Protein kinase-1 (PDK1)

Activated PDK1s lead to cascades causing:

glycogen synthesis

glucose transport

cell growth and differentiation

IR-P controls diverse effects: glucose transport

4) phosphorylation of the APS/Cbl complex

phosphorylation of the APS/Cbl complex

• stimulation of TC10 (monomeric G protein) and (PI3K-independent)

• regulation of glucose transport involving the participation of lipid rafts

Insulin is a hormone that can trigger a variety of physiological effects which makes is not possible to be part of a

• one hormone-one target regulatory system