Question

6. You have discovered a new chemical signaling agent (X) that results in the secretion of compound Z in a specific, dose-dependent fashion. You believe its effects are due to binding a G-protein coupled receptor (GPCR). You have a cell line that expresses a variety of GPCRs. You have access to the following reagents. For each, describe its effects on GPCR mediated cell signaling and how you could use it to determine if your chemical signaling agent operates through a Gs, Gi, or Gq pathway.

dibutyryl cAMP

theophylline

cholera toxin

pertussis toxin

ionomycin

phorbol 12-myristate 13-acetate

Answer 1

Dibutyryl cAMP- it is a type of cAMP analogue responsible for activating cAMP and regulating cAMP dependent protein kinase activation. They are responsible for inhibiting phosphodiestarases. The analogue mimics activation of cAMP, similarly as stimulatory G-protien mediated signalling. since there is continous synthesis of cAMP and regulation of cAMP dependant kinases,thus Dibutyryl cAMP operates through stimulatory G protein (Gs) pathway.

Theophylline- are non-selective inhibitor of phosphodiesterase. The phosphodiestarases are responsible for breaking of cyclin AMP and cyclic GMP. Thus, Theophylline causes increase in concentration of cAMP and cGMP by inhibiting phosphodiestarase and stimulating G-protein for synthesis of cAMP and cGMP. Thus, Theophylline may act through Gs (stimulatory G-protiens).

Cholera Toxin- Cholera toxin is produced by Vibrio cholerae bacterium. Binding of Cholera toxin to GPCRs causes conformational changes in the GPCR due to which the alpha subunit of trimeric G-protein dissociates its bound GDP and binds to GTP,and detaches from beta, gamma subunit, thereby activating the G-protein. The activated G-protein-GTP then binds to adenylate cyclase and activates it. The activated adenylate cyclase then produces cAMP from ATP. The A-promoter of Cholera toxin is an ADP-ribosylating enzyme. The toxin converts NAD+ into ADP-ribose and nicotinamide. The ADP ribose so produced attaches to the G-alpha protein, causing conformational changes in the G-alpha protein. This binding alters its GTPASE activity as a result of which G-alpha loses its ability to hydrolyze the bound GTP to GDP, and remains in stimulatory form producing excess of cAMP. Excess cAMP causes removal of ions from the cell. Change in osmotic pressure, causes water to move with ions into the lumen of the intestinal cells, thereby dehydrating the tissue and causiing diahrroea. Since the cholera toxin keeps the GPCR signaling in stimulatory state by affecting the ability of G-alpha to hydrolyze bound GTP, the Cholera toxin operates through Gs pathway.

Pertussis toxin- is responsible for causing Whooping cough (Bordetella pertusis). Pertusis toxins are a type of ADP ribosylating enzymes, responsible for ribosylating aplha subunit of G-protein (GPCR). this binding prevents the G-aplha protein to unbound its GDP, and interact with GPCR. thus remaining inactive. As the interaction prevents inhibition of adenynyl cyclase leading to excessive production of cAMP. The pertussis toxin operates through Gi pathway.

Ionomycin- Ionomycin is a calcium ionophore, responsible for initiating the hydrolysis of phosphoinositides. Similar to the Gq -GPCR signaling, they are responsible for increaing intracellular calcium ion concentration and activating enzyme PKC. since they operate similarly as GPCR-Gq signalling where Gq protein activates plasma membrane bound enzyme phospholipase C-beta. These PLCs require calcium ions which is provided by synthetic ionophores. hence, the mode of action of ionophore is similar to Gq-GPCRs

Phorbol 12-myristate 13- acetate (PMA)- is a small organic compound which is resposible for desensitization of receptors. PMA, works by directly activating protein kinase C (PKC), via activation of diacyl glycerol (DAG, a component of Gq signalling pathway). PMA mimics the action of DAG and activates PKC which is a serine/ threonine protein kinase responsible for phosphorylating number of proteins involved in tumor progression. PMA is responsible for tumor progression and development. thus, PMA operates through Gq pathway.