The β2 adrenergic receptor (β2AR) has provided a paradigm to elucidate how G protein-coupled receptors (GPCRs) control intracellular signaling, including the discovery that β-arrestins, which bind to ligand-activated GPCRs, are central for GPCR function. We used genome editing, conditional gene deletion, and siRNAs to determine the roles of β-arrestin (β-arr) 1 and 2 in β2AR internalization, trafficking, and signaling to ERK. We found that only β-arr2 was essential for β2AR internalization. Surprisingly, β-arr1 and β-arr2 and receptor internalization were dispensable for ERK activation. Instead, β2AR signaled through Gαs and Gβγ subunits through a pathway that involved the tyrosine kinase SRC, the adaptor protein SHC, the guanine nucleotide exchange factor SOS, the small GTPase RAS, and the kinases RAF and MEK, which led to ERK activation. These findings provide a molecular framework for β2AR signaling through β-arrestin-independent pathways in key physiological functions and pathological conditions.

Excessive FGF23 has been identified as a pivotal phosphaturic factor leading to renal phosphate-wasting, and the subsequent development of rickets and osteomalacia. In contrast, loss of FGF23 in mice (Fgf23−/−) leads to high serum phosphate, calcium and 1,25-vitamin-D levels resulting in early lethality attributable to severe ectopic soft-tissue calcifications and organ-failure. Paradoxically, Fgf23−/− mice exhibit a severe defect in skeletal mineralization despite high levels of systemic mineral ions and abundant ectopic mineralization, an abnormality that remains largely unexplained. Through use of in situ hybridization, immunohistochemistry and immunogold labeling coupled with electron microscopy of bone samples we discovered that expression and accumulation of osteopontin (Opn/OPN) was markedly increased in Fgf23−/− mice. These results were confirmed by qPCR-analyses of Fgf23−/− bones and ELISA measurements of serum OPN. To investigate whether elevated OPN levels were contributing to the bone mineralization defect in Fgf23−/− mice, we generated Fgf23−/−/Opn−/− double-knockout mice (DKO). Biochemical analyses showed that the hypercalcemia and hyperphosphatemia observed in Fgf23−/− mice remained unchanged in DKO mice, however µCT and histomorphometric analyses showed a significant improvement in total mineralized bone-volume. The severe osteoidosis was markedly reduced and a normal mineral apposition rate was present in DKO mice, indicating that increased OPN levels in Fgf23−/− mice are at least in part responsible for the osteomalacia. Moreover, the increased OPN levels were significantly decreased upon lowering serum phosphate by feeding low phosphate diet or deletion NaPi2a, indicating phosphate attributes in part to the high OPN levels in Fgf23−/− mice. In summary, our results suggest that increased OPN is an important pathogenic factor mediating the mineralization defect and the alterations in bone metabolism observed in Fgf23−/− bones.