GPR133 (ADGRD1), an adhesion G-protein-coupled receptor, is necessary for glioblastoma growth.

Glioblastoma (GBM) is a deadly primary brain malignancy with extensive intratumoral hypoxia. Hypoxic regions of GBM contain stem-like cells and are associated with tumor growth and angiogenesis. The molecular mechanisms that regulate tumor growth in hypoxic conditions are incompletely understood. Here, we use primary human tumor biospecimens and cultures to identify GPR133 (ADGRD1), an orphan member of the adhesion family of G-protein-coupled receptors, as a critical regulator of the response to hypoxia and tumor growth in GBM. GPR133 is selectively expressed in CD133+ GBM stem cells (GSCs) and within the hypoxic areas of PPN in human biospecimens. GPR133 mRNA is transcriptionally upregulated by hypoxia in hypoxia-inducible factor 1α (Hif1α)-dependent manner. Genetic inhibition of GPR133 with short hairpin RNA reduces the prevalence of CD133+ GSCs, tumor cell proliferation and tumorsphere formation in vitro. Forskolin rescues the GPR133 knockdown phenotype, suggesting that GPR133 signaling is mediated by cAMP. Implantation of GBM cells with short hairpin RNA-mediated knockdown of GPR133 in the mouse brain markedly reduces tumor xenograft formation and increases host survival. Analysis of the TCGA data shows that GPR133 expression levels are inversely correlated with patient survival. These findings indicate that GPR133 is an important mediator of the hypoxic response in GBM and has significant protumorigenic functions. We propose that GPR133 represents a novel molecular target in GBM and possibly other malignancies where hypoxia is fundamental to pathogenesis.

Mechanism for down-regulation of CD28 by Nef.

SIV and HIV Nef proteins disrupt T-cell receptor machinery by down-modulating cell surface expression of CD4 and expression or signaling of CD3-TCR. Nef also down-modulates class I major histocompatibility complex (MHC) surface expression. We show that SIV and HIV-1 Nefs down-modulate CD28, a major co-stimulatory receptor that mediates effective T-cell activation, by accelerating CD28 endocytosis. The effects of Nef on CD28, CD4, CD3 and class I MHC expression are all genetically separable, indicating that all are selected independently. In cells expressing a Nef-green fluorescent protein (GFP) fusion, CD28 co-localizes with the AP-2 clathrin adaptor and Nef-GFP. Mutations that disrupt Nef interaction with AP-2 disrupt CD28 down-regulation. Furthermore, HIV and SIV Nefs use overlapping but distinct target sites in the membrane-proximal region of the CD28 cytoplasmic domain. Thus, Nef probably induces CD28 endocytosis via the AP-2 pathway, and this involves a ternary complex containing Nef, AP-2 and CD28. The likely consequence of the concerted down-regulation of CD28, CD4 and/or CD3 by Nef is disruption of antigen-specific signaling machineries in infected T cells following a productive antigen recognition event.

Two elements target SIV Nef to the AP-2 clathrin adaptor complex, but only one is required for the induction of CD4 endocytosis.

The simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) Nef proteins induce the endocytosis of CD4 and class I MHC molecules. Here we show that SIV Nef interacts with the AP-2 adaptor complex via two elements located in the N-terminal region of the Nef molecule, but only the N-distal element is required to induce CD4 endocytosis. This N-distal AP-2 targeting element contains no canonical endocytic signals and probably contacts the AP-2 complex via a novel interaction surface. The data support a model where SIV Nef induces CD4 endocytosis by promoting the normal interactions between the di-leucine sorting signal in the CD4 cytoplasmic domain and AP-2, but does not substitute for the CD4-AP-2 adaptor interaction. Neither element is important for the induction of class I MHC endocytosis, thus indicating that different mechanisms underlie the induction of class I MHC and CD4 endocytosis by Nef. In contrast to SIV Nef, HIV-1 Nef interacts with AP-2 via a surface containing a di-leucine endocytosis signal in the C-terminal disordered loop of Nef. The fact that genetic selection maintains similar molecular interactions via different surfaces in SIV and HIV-1 Nef proteins indicates that these interactions have critical roles for the viral life cycle in vivo.