Biology subjects

Stepniewski, T.

Publications and source records attributed to Stepniewski, T..

3 recordsLinked to original sources

bioRxiv2025

An arginine switch drives the stepwise activation of β-arrestin

{beta}-arrestins ({beta}arrs) play a crucial role in regulating G protein-coupled receptor (GPCR) signaling and trafficking. Canonically, interactions of {beta}arr with the phosphorylated intracellular GPCR-tail induce a multi-step conformational transition that results in the activation of {beta}arr. Depending on the specific interaction pattern with the receptor, {beta}arrs adopt multiple conformational states, each tightly linked to a specific functional outcome of {beta}arr recruitment. Despite its physiological relevance, the structural determinants of {beta}arr activation remain poorly understood. Using a combination of molecular dynamics simulations, biochemical and cell-based experiments, we reveal how specific interactions with a prototypical GPCR promote the unbinding of the {beta}arr2 C-tail--a crucial step in arrestin activation. Importantly, we observe that the expulsion of the C-tail is promoted by the displacement of a conserved arginine residue (Arg394) within the {beta}arr polar core, which we dub "the arginine switch." Our study uncovers a previously unknown molecular switch that, upon engagement, destabilizes the polar core as a crucial step in the GPCR-induced {beta}arr activation.

biochemistry↗

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↗

bioRxiv2019

Genetically encoded intrabody sensors illuminate structural and functional diversity in GPCR-β-arrestin complexes

Interaction of {beta}-arrestins ({beta}arrs) upon agonist-stimulation is a hallmark of G protein-coupled receptors (GPCRs) resulting in receptor desensitization, endocytosis and signaling. Although overall functional roles of {beta}arrs are typically believed to be conserved across different receptors, emerging data now clearly unveils receptor-specific functional contribution of {beta}arrs. The underlying mechanism however remains mostly speculative and represents a key missing link in our current understanding of GPCR signaling and regulatory paradigms. Here, we develop synthetic intrabody-based conformational sensors that help us visualize the assembly and trafficking of GPCR-{beta}arr1 complexes in cellular context for a broad set of receptors with spatio-temporal resolution. Surprisingly, these conformational sensors reveal a previously unappreciated level of diversity in GPCR-{beta}arr complexes that extends beyond the current framework of affinity-based classification and phosphorylation-code-based interaction patterns. More importantly, this conformational diversity arising from spatial signature of phosphorylation sites manifests directly in the form of distinct functional outcomes, including even opposite contribution of {beta}arrs in signal-transduction for different receptors. Taken together, these findings uncover that despite an overall similar interaction and trafficking patterns; critical structural and functional differences exist in {beta}arr complexes for different GPCRs that define and fine-tune receptor-specific downstream responses.

cell biology↗