ABSTRACT
While the existence and functional role of class C G-protein-coupled receptors (GPCR) dimers is well established, there is still a lack of consensus regarding class A and B GPCR multimerization. This lack of consensus is largely due to the inherent challenges of demonstrating the presence of multimeric receptor complexes in a physiologically relevant cellular context. The C-X-C motif chemokine receptor 4 (CXCR4) is a class A GPCR that is a promising target of anticancer therapy. Here, we investigated the potential of CXCR4 to form multimeric complexes with other GPCRs and characterized the relative size of the complexes in a live-cell environment. Using a bimolecular fluorescence complementation (BiFC) assay, we identified the ß2 adrenergic receptor (ß2AR) as an interaction partner. To investigate the molecular scale details of CXCR4-ß2AR interactions, we used a time-resolved fluorescence spectroscopy method called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS can resolve membrane protein density, diffusion, and multimerization state in live cells at physiological expression levels. We probed CXCR4 and ß2AR homo- and heteromultimerization in model cell lines and found that CXCR4 assembles into multimeric complexes larger than dimers in MDA-MB-231 human breast cancer cells and in HCC4006 human lung cancer cells. We also found that ß2AR associates with CXCR4 multimers in MDA-MB-231 and HCC4006 cells to a higher degree than in COS-7 and CHO cells and in a ligand-dependent manner. These results suggest that CXCR4-ß2AR heteromers are present in human cancer cells and that GPCR multimerization is significantly affected by the plasma membrane environment.
Subject(s)
Neoplasms , Receptors, Adrenergic, beta-2 , Receptors, CXCR4 , Signal Transduction , Animals , Cricetinae , Humans , CHO Cells , Cricetulus , Membrane Proteins/metabolism , Neoplasms/metabolism , Receptors, CXCR4/metabolism , Receptors, Adrenergic, beta-2/metabolism , Protein MultimerizationABSTRACT
O-GlcNAc transferase (OGT) is an enzyme that catalyzes the O-GlcNAc modification of nucleocytoplasmic proteins and is highly expressed in many types of cancer. However, the mechanism regulating its expression in cancer cells is not well understood. This study shows that OGT is a substrate of the E3 ubiquitin ligase X-linked inhibitor of apoptosis (XIAP) which plays an important role in cancer pathogenesis. Although LSD2 histone demethylase has already been reported as an E3 ubiquitin ligase in lung cancer cells, we identified XIAP as the main E3 ubiquitin ligase in colon cancer cells. Interestingly, OGT catalyzes the O-GlcNAc modification of XIAP at serine 406 and this modification is required for the E3 ubiquitin ligase activity of XIAP toward specifically OGT. Moreover, O-GlcNAcylation of XIAP suppresses colon cancer cell growth and invasion by promoting the proteasomal degradation of OGT. Therefore, our findings regarding the reciprocal regulation of OGT and XIAP provide a novel molecular mechanism for controlling cancer growth and invasion regulated by OGT and O-GlcNAc modification.
Subject(s)
Colonic Neoplasms/metabolism , N-Acetylglucosaminyltransferases/metabolism , X-Linked Inhibitor of Apoptosis Protein/metabolism , Cell Line, Tumor , Cell Proliferation/physiology , Colonic Neoplasms/pathology , Glycosylation , HCT116 Cells , HEK293 Cells , Humans , Neoplasm Invasiveness , Transfection , UbiquitinationABSTRACT
Unlike other types of glycosylation, O-GlcNAcylation is a single glycosylation which occurs exclusively in the nucleus and cytosol. O-GlcNAcylation underlie metabolic diseases, including diabetes and obesity. Furthermore, O-GlcNAcylation affects different oncogenic processes such as osteoblast differentiation, adipogenesis and hematopoiesis. Emerging evidence suggests that skeletal muscle differentiation is also regulated by O-GlcNAcylation, but the detailed molecular mechanism has not been fully elucidated. In this study, we showed that hyper-O-GlcNAcylation reduced the expression of myogenin, a transcription factor critical for terminal muscle development, in C2C12 myoblasts differentiation by O-GlcNAcylation on Thr9 of myocyte-specific enhancer factor 2c. Furthermore, we showed that O-GlcNAcylation on Mef2c inhibited its DNA binding affinity to myogenin promoter. Taken together, we demonstrated that hyper-O-GlcNAcylation attenuates skeletal muscle differentiation by increased O-GlcNAcylation on Mef2c, which downregulates its DNA binding affinity.
Subject(s)
Acetylglucosamine/metabolism , Cell Differentiation , Muscle Development , Myoblasts/cytology , Acylation , Animals , Cell Line , Glycosylation , HEK293 Cells , Humans , MEF2 Transcription Factors/metabolism , Mice , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Myoblasts/metabolismABSTRACT
Nucleocytoplasmic O-GlcNAc transferase (OGT) attaches a single GlcNAc to hydroxyl groups of serine and threonine residues. Although the cellular localisation of OGT is important to regulate a variety of cellular processes, the molecular mechanisms regulating the nuclear localisation of OGT is unclear. Here, we characterised three amino acids (DFP; residues 451-453) as the nuclear localisation signal of OGT and demonstrated that this motif mediated the nuclear import of non-diffusible ß-galactosidase. OGT bound the importin α5 protein, and this association was abolished when the DFP motif of OGT was mutated or deleted. We also revealed that O-GlcNAcylation of Ser389, which resides in the tetratricopeptide repeats, plays an important role in the nuclear localisation of OGT. Our findings may explain how OGT, which possesses a NLS, exists in the nucleus and cytosol simultaneously.
