J. Daniel Jensen and Dr. Laura Bridgewater, Microbiology and Molecular Biology
GDF5, also known as cartilage-derived morphogenetic protein-1 (CDMP1), is a member of the transforming growth factor beta (TGFβ) protein superfamily. TGFβ proteins play an integral role in cell proliferation and differentiation, matrix synthesis, and apoptosis. First identified in 1981, highly-conserved TGFβ protein homologues have since been identified in a wide range of animal species, a compelling indication of their importance in biological function.
Translation of the GDF5 mRNA transcript results in a primary sequence 501 amino acids in length. This primary sequence is composed of an N-terminal propeptide region (1–381) and a mature peptide chain (382–501). The preprocessed GDF5 is cleaved to mature GDF5 at the polybasic (378) RRKR cleavage site by both furin and subtilisin-like proprotein convertase-6 (SPC6). Further post-translational modifications (homodimerization of two GDF5 mature peptides by disulfide linkage and C-terminus glycoslylation) occur in the endoplasmic reticulum (ER). The dimerized, processed GDF5 protein is then secreted into the ECM through normal cellular mechanisms.
Recently in our lab, both bone morphogenetic protein-2 (BMP2) and bone morphogenetic protein-4 (BMP4), also members of the TGFβ superfamily and very similar in structure to GDF5, had been shown to be present in the nucleus through in vivo immunofluorescence microscopy. These findings are significant because neither protein had been observed in the nucleus previously. Evidence suggested that the same would be true of GDF5 as well, despite it having not been observed in the nucleus, either. Aside from their similar structures and functions, one of the many compelling indications that GDF5 would behave similarly to BMP2 and BMP4 is that GDF5 had several possible nuclear localization signals (NLS) located throughout its primary amino acid sequence.
NLS sequences generally possess a short stretch of positively charged amino acids, such as arginine and lysine. A variation on the NLS motif is the bipartite NLS, which consists of two groupings of basic amino acids, separated by a spacer of approximately 10 amino acids. These positively charged amino acid sequences are recognized by and bind to cytosolic nuclear transport receptors, such as importin, and are subsequently translocated into the nucleus through the nuclear pore complex. A computer-based predictive modeling program (PSORTII, http://psort.nibb.ac.jp/form2.html) was used to predict possible NLS sequences in GDF5 based on the positions of basic amino acid residues. Three NLS sequences were identified in the propeptide region of GDF5 (215-PVVRKQR, 243-RKKP, and 377-RRKRR), while a bipartite NLS was found to span the site of proteolytic cleavage (378-RKRR↕APLATRQGKRPSK, where the double arrow marks the cleavage site). Further investigation revealed that the putative bipartite NLS spanning the proteolytic cleavage site was homologous with, but not identical to, the functional NLSs that were recently discovered in BMP2 and BMP4. The particular location of this sequence suggests that an uncleaved variant of GDF5 may be localized to the nucleus. A functional NLS at this location would be disrupted if cleaved, resulting in the eventual transport of GDF5 to the ECM. If left uncleaved, however, the NLS would retain functionality, thus localizing GDF5 to the nucleus.
The goals of my project were to determine: 1) if endogenous GDF5 is found in the nucleus of mammalian cell lines, and 2) if any of the aforementioned NLSs are responsible for directing nuclear localization. Rat chondrosarcoma (RCS) cells were used in these experiments because GDF5 was first identified as a possible DNA-binding protein on the enhancer region of a chondrocyte-specific gene, which suggests that it may be present in RCS nuclei. Mouse mesenchyme fibroblasts (10T1/2), which are less differentiated than RCS cells, were also used as a second cell line to determine whether experimental results were specific to chondrocytes, or demonstrated a more general trend.
Immunohistochemical staining of GDF5 in fixed cells, and subsequent visualization using laser confocal microscopy, were used to demonstrate the presence of naturally occurring, endogenous GDF5 in the nucleus in vivo. (Figure 1) These results demonstrate that endogenous GDF5 is localized to the nucleus. Similar experiments with other mammalian cell lines support and strengthen this conclusion.
Furthermore, fusion constructs containing sequences that coded for GFP-PVVRKQR, GFP-RKKP and GFP-RRKRR were introduced into RCS and 10T1/2 cells through transfection. These constructs expressed GFP protein that was localized to the cytoplasm of RCS cells. This result suggested that these three putative NLS sequences are not functional, and cannot direct the nuclear localization. The bipartite NLS was also fused to GFP and transfected into RCS cells. GFP-bipartite NLS, however, directed GFP to the nucleus in both RCS and 10T1/2 cells. (Figure 2) The functionality of the bipartite NLS in the GFP/NLS fusion construct suggests that the bipartite NLS is responsible for directing nuclear localization of endogenous GDF5 in RCS cells.