Correlation between leucine rich domain and the stability of LRWD1 protein in human NT2/D1 cells

Purpose: LRWD1 is a protein that contains LRR and WDs domains and is important in regulating spermatogenesis. However, the roles of LRR or WDs domains in the expression of LRWD1 remain unclear. Materials and methods: The NT2/D1 cells separately transfected with full length of LRWD1 gene (LRWD^WT) or genes with deleted sequences in the LRR domain (LRWD1^ΔLRR), WD1 domain (LRWD1^ΔWD1), WD2 domain (LRWD1^ΔWD2), WD3 domain (LRWD1^ΔWD3) and entire three WD domains (LRWD1^Δ3xWD) were applied to investigate the expression levels of LRWD1 protein by either Western blot or flow cytometry. The associated proteins in these mutated LRWD1 proteins were identified by mass spectrometry. Results: Deletion of the LRR domain significantly decreased the expression of LRWD1 protein. With the treatment of MG132, the LRR domain may functions in preventing LRWD1 protein from proteasome-mediated degradation. In the co-immunoprecipitation analysis, protein receptor of tumor necrosis factor 2 (TNFR2) was specifically observed to be associated with LRR-deficient LRWD1 protein. Conclusions: The LRR domain is significantly correlated to the stability of LRWD1 protein. Determining if the stability is modulated by TNFR2 is worthy of further study.