ROY ELISA (BBSH18096548)

1. GPCRs are the most common and abundant class of receptors in most eukaryotes. These
receptors are integral membrane protein having a common 7 transmembrane segment
structure. GPCRs are characterized by an extracellular N-terminus,
seven transmembrane helices connected by three intracellular and three extracellular loops,
and lastly an intracellular C-terminus. Once a ligand binds on the ligand-binding domain
formed by amino acids located at either interior or exterior membrane, the receptor is
activated which in turn activates the G-protein followed by interaction with downstream
signal transduction proteins. These GPCRs interact with the trimeric G proteins in the C3 and
C4 cytoplasmic regions.

2.

Step 1: Ligand (hormone) binds to GPCR triggering conformational change in the receptor
shape
Step 2: Conformational change leads promoting the binding of hormone to the G protein.
Step 3: GDP dissociation triggered by binding to the activated GPCR
Step 4: GTP binds to activated G-protein (Ga) thereby triggering dissociation of Ga both from
receptor and Gβγ
Step 5: The hormone dissociates from the receptor and GTP- G a complex binds to the effector
protein thereby regulating its activity.
 ROY ELISA (BBSH18096548)

Step 6: The Ga dissociates from the effector due to the hydrolysis of GTP to GDP. The free
Ga binds back to the Gβγ, thereby forming back the trimeric G=protein.

3. Activated G-proteins (Ga) target on adenyl cyclase by which the adenyl cyclase is activated
by the GTP-bound α subunit. This activation of adenyl cyclase helps in catalyzing the ATP
molecules to synthesize cAMP. Various cells express various types of GPCRs that all bind to
adenyl cyclase to synthesize cAMP. The net activity of this adenyl cyclase is dependent on
the collective G protein signaling levels through multiple GPCRs.
Ligands can also bind to GPCRs activating an enzyme called, phospholipase C in the cytosol.
Phospholipids bound to the cell membrane such as phosphatidylinositol 4,5-bisphosphate
(PIP2) are cleaved into, diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). The
DAG molecule activates PKC by activating a phosphorylation cascade and phosphorylate
certain proteins, whereas the IP3 molecule binds to a receptor on a ligand gated Ca 2+ channel
on the outer surface of smooth endoplasmic reticulum and triggers the release of Ca 2+ into the
cytosol of the cell. Both IP3 and DAG acts as second messengers.

4. Nitric oxide (NO) stimulate the production of cGMP by activating the enzyme guanylate
cyclase to convert GTP into cGMP. The NO/cGMP pathway is responsible for regulating the
concentration of free Ca2+ in the smooth muscles. The cGMP has an effect on the cells
which is mediated with the help of protein kinase G (PKG) activation. Once PKG is activated
by cGMP, an enzyme- myosin phosphatase is activated which in turn releases Ca2+ from the
intercellular stores in the smooth muscle cells. These Ca2+ are responsible for the production
of NO synthase that helps in the synthesis of NO from arginine. This NO gas then diffuses
into the smooth muscle cells and cause vasodilation by activating more guanyl cyclase and
increase the cGMP concentration. Due to the vasodilation properties of NO, certain drugs
such as nitroglycerin are administered to patients who have angina or being treated after
myocardial infarction.

5. With an increase in Ca2+ concentrations, the PLA2 is activated. As ligand binds to its
receptor, it induces the activation of PLC by which the PLC cleaves off into DAG and IP3
thereby opening the Ca2+ channels in the endoplasmic reticulum. Phosphorylation of cPLA2
by mitogen-activated protein kinases (MAPK) also play a role in activation. The activated
PLA2 then translocate to the membranes of Golgi apparatus, endoplasmic reticulum and/or
the nucleus from which arachidonate is released thereby triggering the arachidonic acid
pathway. The final product prostaglandins are lipids deriving arachidonate with the help of
COX enzyme actions which promotes inflammation in the body. Prostaglandins play a very
important role in generating an inflammatory response contributing to the development of
acute inflammation.