KLHL12 promotes non-lysine ubiquitination of the dopamine receptors D-4.2 and D-4.4, but not of the ADHD-associated D-4.7 variant

Dopamine D-4 Receptor Polymorphism : The dopamine D-4 receptor has an important polymorphism in its third intracellular loop that is intensively studied and has been associated with several abnormal conditions, among others, attention deficit hyperactivity disorder. KLHL12 Promotes Ubiquitination of the Dopamine D-4 Receptor on Non-Lysine Residues : In previous studies we have shown that KLHL12, a BTB-Kelch protein, specifically interacts with the polymorphic repeats of the dopamine D-4 receptor and enhances its ubiquitination, which, however, has no influence on receptor degradation. In this study we provide evidence that KLHL12 promotes ubiquitination of the dopamine D-4 receptor on non-lysine residues. By using lysine-deficient receptor mutants and chemical approaches we concluded that ubiquitination on cysteine, serine and/or threonine is possible. Differential Ubiquitination of the Dopamine D-4 Receptor Polymorphic Variants : Additionally, we show that the dopamine D-4.7 receptor variant, which is associated with a predisposition to develop attention deficient hyperactivity disorder, is differentially ubiquitinated compared to the other common receptor variants D-4.2 and D-4.4. Together, our study suggests that GPCR ubiquitination is a complex and variable process

Novel strategy for rapid functional in vivo validation of oncogenic drivers in haematological malignancies

In cancer research, it remains challenging to functionally validate putative novel oncogenic drivers and to establish relevant preclinical models for evaluation of novel therapeutic strategies. Here, we describe an optimized and efficient pipeline for the generation of novel conditional overexpression mouse models in which putative oncogenes, along with an eGFP/Luciferase dual reporter, are expressed from the endogenous ROSA26 (R26) promoter. The efficiency of this approach was demonstrated by the generation and validation of novel R26 knock-in (KI) mice that allow conditional overexpression of Jarid2, Runx2, MN1 and a dominant negative allele of ETV6. As proof of concept, we confirm that MN1 overexpression in the hematopoietic lineage is sufficient to drive myeloid leukemia. In addition, we show that T-cell specific activation of MN1 in combination with loss of Pten increases tumour penetrance and stimulates the formation of Lyl1(+) murine T-cell lymphoblastic leukemias or lymphomas (T-ALL/T-LBL). Finally, we demonstrate that these luciferase-positive murine AML and T-ALL/T-LBL cells are transplantable into immunocompromised mice allowing preclinical evaluation of novel antileukemic drugs in vivo

Treatment with a highly reducing agent decreases ubiquitination of D_4.2R WT and D_{4.2 4KR}R mutant.

<p>HEK293T cells were transiently transfected as indicated. 48 h post-transfection, cells were harvested and lysed. 5% of the lysates were used for IB to visualize (Flag Ub)n-proteins, HA D₄R, and Etag KLHL12, respectively (left panels). The rest of the lysates were subjected to IP with anti-HA (16B12). After IP proteins were eluted at 95°C with or without addition of 100 mM DTT. Next, a second round of IP with anti-HA antibody was performed. Specific purification of the receptor after the second IP was confirmed upon IB with rat anti-HA (1:2000), whereas receptor ubiquitination was revealed upon IB with anti-Flag-HRP (right panels, 1:2000). Shown are results from a single experiment, representative of three independent experiments.</p

All common D₄R polymorphic variants interact with KLHL12 while D_4.0R, lacking repeats in the third intracellular loop, does not interact.

<p>HEK293T cells were transiently transfected as indicated. 48 h post-transfection, cells were harvested and lysed. Part of the lysate was used for IB to verify expression of HA-tagged D₄R variants and Etag KLHL12 (left panels). The rest of the lysate was subjected to IP with anti-HA (16B12). Specific purification of the receptor after IP was confirmed upon IB with rat anti-HA (1:2000). Interaction of Etag KLHL12 with the D₄R polymorphic variants was verified by IB with anti-Etag (1:2000). Data shown are representative of three independent experiments. Expected molecular weights of different D₄R variants: D_4.0R (44–50 kDa), D_4.2R (47-53kDa), D_4.4R (51–57 kDa), D_4.7R (56–62 kDa).</p