Construction of G Alpha-16 Chimeras for Detection of GPCR Activation

Abstract

G protein-coupled Receptors (GPCRs) transduce various extracellular stimuli to intracellular signaling across the plasma membrane. Ligand binding causes a conformational change of the GPCR, resulting in interaction with the GDP-bound G alpha, which forms a heterotrimer with G beta-gamma. Nucleotide exchange of GDP for GTP follows causing the GTP-bound active G alpha to dissociate from G beta-gamma so that both subunits can transduce signaling. There are four subclasses of G alpha subunits, such as G alpha-s, G alpha-i, G alpha-q, and G alpha-12/13, which activate distinct effectors. Members of the G alpha-q subclass (G alpha-q, G alpha-11, G alpha-14, and G alpha-16) directly bind and stimulate the catalytic activity of phospholipase C beta isozymes, which produce two second messengers, diacylglycerol and inositol 1,4,5-triphosphate (IP3) from phosphatidylinositol-(4,5)-bisphosphate. IP3 induces calcium release from the endoplasmic reticulum (ER).In order to detect GPCR-G protein coupling by monitoring calcium mobilization, we constructed chimeric G proteins. Calcium mobilization, elicited by GPCR stimulation, was monitored using the calcium indicator Fluo-4-AM. It is known that the C-terminal sequences of G alpha play critical roles in interactions with GPCRs. Because G alpha-16 displays promiscuity toward GPCRs, C-terminal residues (11 or 30 amino acids) of G alpha-16 were replaced with those of other subclasses of G alpha. Human embryonic kidney (HEK) 293 cells were transfected with GPCRs and G alpha-16 chimeras (G alpha-16/s, G alpha-16/o, G alpha-16/i1-i2, G alpha-16/i3, G alpha-16/13). Other assays are also utilized as controls of chimeric G alpha-16 signaling. Additionally, G alpha-16 chimera constructs are being applied to detect modulated- or biased-G protein coupling by heteromerization of GPCRs.