Question

Inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) are second messenger molecules derived from the cleavage of the phosphatidylinositol-4,5-bisphosphate (PIP2) by activated phospholipase C.

A) Describe the role of IP3 in causing a rise in cytosolic Ca2+ concentration.
B)How do cells restore resting levels of cytosolic Ca+2?
C)What is the principal function of DAG?

Answer 1

Phospholipase C is phosphodiesterase enzyme involved in hydrolysis of phosphatidylinositol 4, 5–bisphosphate (PI 4, 5–P2), upon activation by binding of ligand to receptor. PIP2, that is present in the plasma membrane is cleaved to secondary messenger inositol 1,4,5–trisphosphate (IP3) and diacylglycerol (DAG). IP3 is released into the cytoplasm while DAG remains attached to plasma membrane.

A) IP3 is a small polar molecule that is involved in release of calcium from their intracellular stores in the cell. Normally, calcium levels in cytoplasm are maintained at low levels in cytoplasm in the unstimulated state. This low level of calcium is due to calcium pumps. The calcium pumps will export calcium ions out of the cell by the plasma membrane. Calcium is also pumped into the endoplasmic reticulum, thereby allowing calcium ions to be stored in the ER. ER has active Ca2+ transport of calcium ions by transfer by sarco/endoplasmic reticulum Ca2+ ATPases (SERCA). IP3, once it is released by action of phospholipase C will bind to its receptors on the ER (or other intracellular calcium storage organelle). These IP3 receptors are ligand gated calcium channels. IP3 binds to the tetrameric receptor between the amino acid 226 and 578. The binding will cause a conformational change in the receptor, allowing the ion channels to be opened. At cytoplasmic calcium concentrations below 500 nM, the IP3 receptor is activated by binding to IP3. However, when the calcium levels in cytoplasm rise above 500 nM, there is inhibition of the IP3 receptor and the ion channel remain closed. Thus, IP3 increases cytosolic calcium levels.

B) Calcium is an allosteric modulator of IP3 receptor. When there are high levels of cytoplasmic calcium, calcium binds to the IP3 receptor and will inhibit the opening of the calcium channel of the IP3 receptor. This will inhibit the release of calcium from ER. Further, ER has the SERCA pump, which is a calcium ATPase. It uses the energy from ATP hydrolysis to transport cytoplasmic calcium into the ER. Different types of SERCA are expressed in different tissues. When the cytoplasmic calcium increases, calcium will dissociate from EF hands. This allows proteins such as STIM to assemble at the plasma membrane. STIM1 oligomerizes and binds to SOCE channels of the Orai protein. This binding will stimulated calcium influx into intracellular calcium stores.

Calcium also is pumped out of the cell from the plasma membrane via pumps such as PMCA and NCX into the extracellular milieu. SPCA channels on the Golgi membrane also cause transport of cytosolic calcium into the Golgi intracellular stores. All these mechanisms will restore resting low levels of calcium in the cell.

C) Diacylglycerol is a small molecule that remains behind in the plasma membrane when it’s formed by actions of Phospholipase C. formation of DAG will cause increased translocation of protein kinase C to the plasma membrane from the cytosol. DAG can bind directly to protein kinase C at the plasma membrane. However, calcium and phosphatidyl serine are also required for this action of DAG. The binding or DAG occurs at the cysteine rich domain of the N terminal of protein kinase C (PKC). DAG binding to PKC increases its affinity for calcium. This will result in increased activity of PKC. PKC will then phosphorylate downstream protein on their serine and threonine residues. Thus, major function of DAG is to activate protein kinase C for activation of downstream signaling.