Conditionally expressed G alpha 15 couples to endogenous receptors in GH3 cells

Area of Science:

• Cellular signaling
• G protein-coupled receptors
• Biochemistry

Background:

• Mammalian G proteins G15 and G16 are known to couple various receptors to phospholipase C (PLC).
• These G proteins have been proposed as tools for agonist-screening systems.
• Understanding their specific coupling and downstream effects is crucial for drug discovery.

Purpose of the Study:

• To investigate the coupling of endogenous receptors to Galpha15 and Galpha16 in a physiologically relevant system.
• To compare the functional promiscuity of Galpha15 and Galpha16 in response to receptor activation.
• To determine if Galpha15-mediated signaling can be utilized for detecting receptor activation in screening assays.

Main Methods:

• Generation of rat pituitary GH3 cell clones expressing Galpha15 and Galpha16 using a reversed tetracycline-controlled transactivation system.
• Measurement of inositol phosphate production and cytosolic calcium ([Ca2+]i) levels upon receptor activation.
• Assessment of protein kinase C activity and cellular secretion in response to agonists.

Main Results:

• Conditional expression of Galpha15 enhanced agonist-dependent inositol phosphate production from endogenous receptors.
• Galpha16 expression had no significant effect on agonist-dependent PLC activity.
• Galpha15 activation led to protein kinase C stimulation and altered carbachol-induced secretion, unlike Galpha16.
• Galpha15 demonstrated higher functional promiscuity than Galpha16 in this system.

Conclusions:

• Galpha15 exhibits greater functional promiscuity than Galpha16 in coupling endogenous receptors to PLC.
• Galpha15-mediated signaling pathways can modify cellular responses to receptor agonists.
• These findings support the utility of Galpha15 in developing novel receptor screening systems.