Antagonist minigenes identify genes regulated by parathyroid hormone through G protein-selective and G protein co-regulated mechanisms in osteoblastic cells

Parathyroid hormone (PTH) is the major hormone regulating bone remodeling. Binding of PTH to the PTH1 receptor (PTH1R), a heterotrimeric G protein coupled receptor (GPCR), can potentially trigger multiple signal transduction pathways mediated through several different G proteins. In this study, we employed G protein antagonist minigenes inhibiting Gα_s, Gα_q or Gα₁₂ to selectively dissect out which of these G proteins were responsible for effects of PTH(1-34) in targeted signaling and osteogenesis arrays consisting of 159 genes. Among the 32 genes significantly regulated by 24h PTH treatment in UMR-106 osteoblastic cells, 9 genes were exclusively regulated through G_s, 6 genes were solely mediated through G_q, and 3 genes were only controlled through G₁₂. Such findings support the concept that there is some absolute specificity in downstream responses initiated at the G protein level following binding of PTH to the PTH1R. On the other hand, 6 PTH-regulated genes were regulated by both G_s and G_q, 3 genes were regulated by both G_s and G₁₂, and 3 genes were controlled by G_s, G_q and G₁₂. These findings indicate potential overlapping or sequential interactions among different G protein-mediated pathways. In addition, two PTH-regulated genes were not regulated through any of the G proteins examined, suggesting that additional signaling mechanisms may be involved. Selectivity was largely maintained over a 2-48-hour time period. The minigene effects were mimicked by downstream inhibitors. The dissection of the differential effects of multiple G protein pathways on gene regulation provides a more complete understanding of PTH signaling in osteoblastic cells.