BTB Protein KLHL12 targets the dopamine D4 receptor for ubiquitination by a Cul3-based E3 ligase

Dopamine receptors belong to the superfamily of G-protein-coupled receptors and are subdivided into D1-type (D1 and D5) and D2-type (D2, D3, and D4) receptors. The D4 receptor has a remarkable polymorphism in its third intracellular loop, which is under intensive investigation and which has been associated with, among other conditions, attention deficit hyperactivity disorder. Here, we demonstrate that KLHL12, a BTB-Kelch protein, specifically binds to this polymorphic region of the D4 receptor through its Kelch domain. Moreover, we show that KLHL12 also interacts with Cullin3 and thereby functions as an adaptor to target the D4 receptor to an E3 ubiquitin ligase complex. By ubiquitination assays in eukaryotic cells, we further demonstrate that overexpression of KLHL12 strongly promotes ubiquitination of the D4 receptor. In addition, we show that also other dopamine receptor subtypes undergo basal ubiquitination, but this is not affected by KLHL12. These data are the first to show ubiquitination of dopamine receptors and the first to identify a protein specifically interacting with the D4 polymorphism, thereby building up an E3 ligase complex with substrate specificity toward the D4 receptor.     

Biochemical and pharmacological study of N-linked glycosylation of the human serotonin 5-HT₇a receptor

The 5-hydroxytryptamine (5-HT)(7(a)) receptor is a G-protein-coupled receptor critically involved in human psychiatric and neurological disorders. In the present study, we evaluate the presence and the functional role of N-glycosylation of the human 5-HT(7) receptor. Western blot analysis of HEK293T cells transiently expressing the 5-HT(7(a)) receptor in the presence of tunicamycin gave rise to a band shift, indicating the existence of an N-glycosylated form of the 5-HT(7(a)) receptor. To further investigate this, we mutated the two predicted N-glycosylation sites (N5Q and N66Q) and compared the molecular mass of the immunoreactive bands with those of the wild-type receptor, indicating that both asparagines were N-glycosylated. The mutant receptors had the same binding affinity for [(3) H]5-CT and the same potency and efficacy with regard to 5-HT-induced activation of adenylyl cyclase. However, there was a reduction in maximal ligand binding for the single and double mutants compared to the wild-type receptor. Next, membrane labelling and immunocytochemical studies demonstrated that the N-glycosylation mutants were expressed at the cell surface. We conclude that N-glycosylation is not important for cell surface expression of the 5-HT(7) receptor.     

Localization of the gene for sclerosteosis to the van Buchem disease-gene region on chromosome 17q12-q21.

Sclerosteosis is an uncommon, autosomal recessive, progressive, sclerosing, bone dysplasia characterized by generalized osteosclerosis and hyperostosis of the skeleton, affecting mainly the skull and mandible. In most patients this causes facial paralysis and hearing loss. Other features are gigantism and hand abnormalities. In the present study, linkage analysis in two consanguineous families with sclerosteosis resulted in the assignment of the sclerosteosis gene to chromosome 17q12-q21. This region was analyzed because of the recent assignment to this chromosomal region of the gene causing van Buchem disease, a rare autosomal recessive condition with a hyperostosis similar to sclerosteosis. Because of the clinical similarities between sclerosteosis and van Buchem disease, it has previously been suggested that both conditions might be caused by mutations in the same gene. Our study now provides genetic evidence for this hypothesis.