Systematic analysis of the RGS2 degron reveals characteristics of substrate recognition by the F-box protein FBXO44

Harrison J McNabb; Eugene Cho; Mary Pitman; Phillip S Rushton; David Mobley; Benita Sjögren

doi:10.1016/j.jbc.2025.110757

Systematic analysis of the RGS2 degron reveals characteristics of substrate recognition by the F-box protein FBXO44

J Biol Chem. 2025 Nov;301(11):110757. doi: 10.1016/j.jbc.2025.110757. Epub 2025 Sep 22.

Authors

Harrison J McNabb¹, Eugene Cho², Mary Pitman³, Phillip S Rushton¹, David Mobley³, Benita Sjögren⁴

Affiliations

¹ Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana, USA.
² Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, California, USA; Robert A. Mah Molecular Innovation Center, University of California, Irvine, Irvine, California, USA.
³ Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, California, USA.
⁴ Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana, USA; Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, California, USA; Robert A. Mah Molecular Innovation Center, University of California, Irvine, Irvine, California, USA; Department of Biological Chemistry, University of California, Irvine, Irvine, California, USA. Electronic address: jsjogren@uci.edu.

Abstract

Regulator of G protein signaling 2 (RGS2) negatively modulates signaling downstream of G protein-coupled receptors by accelerating GTP hydrolysis at Gα subunits of heterotrimeric G proteins. Decreased RGS2 levels are implicated in numerous diseases, including cardiovascular disease and asthma. Thus, identifying selective means of enhancing RGS2 protein levels would be a viable therapeutic strategy. RGS2 is rapidly degraded through the ubiquitin-proteasomal pathway, and we previously identified F-box only protein 44 (FBXO44) as the substrate recognition component of the E3 ligase responsible for facilitating RGS2 degradation. As such, the RGS2-FBXO44 interaction is a potential target for pharmacological intervention. Detailed information on the FBXO44 recognition site (degron) in RGS2 will aid in structure-based small-molecule inhibitor design, as well as in identifying additional FBXO44 targets, which would help predict possible side effects of targeting this interaction. Thus, the goal of this study was to dissect the molecular properties for FBXO44 binding of the RGS2 degron. We used a peptide array utilizing systematic residue substitution, combined with AlphaFold modeling and molecular dynamics simulations, to identify several amino acid changes that altered binding both positively and negatively. Finally, we experimentally confirmed our results in cells through coimmunoprecipitation and proteasomal inhibition, using full-length RGS2. Altogether, these results provide structural insights into RGS2-FBXO44 binding, which will aid in structure-guided drug discovery efforts. It also provides a framework for building a consensus recognition motif for FBXO44, which could aid in identifying more substrates for this understudied F-box protein.

Keywords: E3 ubiquitin ligase; molecular modeling; peptide array; protein degradation; regulator of G protein signaling.

MeSH terms

Binding Sites
Degrons
F-Box Proteins* / chemistry
F-Box Proteins* / genetics
F-Box Proteins* / metabolism
HEK293 Cells
Humans
Protein Binding
Proteolysis*
RGS Proteins* / chemistry
RGS Proteins* / genetics
RGS Proteins* / metabolism
Substrate Specificity

Substances

RGS Proteins
F-Box Proteins
RGS2 protein, human