Luttrell, L. M. | Search bioRxiv

bioRxiv2019

Conformational sensors and domain-swapping reveal structural and functional differences between β-arrestin isoforms

Desensitization, signaling and trafficking of G protein-coupled receptors (GPCRs) are critically regulated by multifunctional adaptor proteins, {beta}-arrestins ({beta}arrs). The two isoforms of {beta}arrs ({beta}arr1 and 2) share a high degree of sequence and structural similarity, still however, they often mediate distinct functional outcomes in the context of GPCR signaling and regulation. A mechanistic basis for such a functional divergence of {beta}arr isoforms is still lacking. Using a set of complementary approaches including antibody fragment based conformational sensors, we discover structural differences between {beta}arr1 and 2 upon their interaction with activated and phosphorylated receptors. Interestingly, domain swapped chimeras of {beta}arrs display robust complementation in functional assays thereby, linking the structural differences between the receptor-bound {beta}arr1 and 2 with their divergent functional outcomes. Our findings reveal important insights into the ability of {beta}arr isoforms to drive distinct functional outcomes, and underscore the importance of integrating this aspect in the current framework of biased agonism.

biophysics↗

bioRxiv2018

Transcriptomic characterization of signaling pathways associated with osteoblastic differentiation of MC-3T3E1 cells

Bone remodeling involves the coordinated actions of osteoclasts, which resorb the calcified bony matrix, and osteoblasts, which refill erosion pits created by osteoclasts to restore skeletal integrity and adapt to changes in mechanical load. Osteoblasts are derived from pluripotent mesenchymal stem cell precursors, which undergo differentiation under the influence of a host of local and environmental cues. To characterize the autocrine/paracrine signaling networks associated with osteoblast maturation and function, we performed gene network analysis using complementary \"agnostic\" DNA microarray and \"targeted\" NanoString nCounter datasets derived from murine MC3T3-E1 cells induced to undergo synchronized osteoblastic differentiation in vitro. Pairwise datasets representing changes in gene expression associated with growth arrest (day 2 to 5 in culture), differentiation (day 5 to 10 in culture), and osteoblast maturation (day 10 to 28 in culture) were analyzed using Ingenuity Systems Pathways Analysis to generate predictions about signaling pathway activity based on the temporal sequence of changes in target gene expression. Our data indicate that some pathways known to be involved in osteoblast differentiation, e.g. Wnt/{beta}-catenin signaling, are most active early in the process, while others, e.g. TGF{beta}/BMP, cytokine/JAK-STAT and TNF/RANKL signaling, increase in activity as differentiation progresses. Collectively, these pathways contribute to the sequential expression of genes involved in the synthesis and mineralization of extracellular matrix. These results provide insight into the temporal coordination and complex interplay between signaling networks controlling gene expression during osteoblast differentiation. A more complete understanding of these processes may aid the discovery of novel methods to promote osteoblast development for the treaent of conditions characterized by low bone mineral density.

cell biology↗