Evidence that IGF binding protein-5 functions as a ligand-independent transcriptional regulator in vascular smooth muscle cells.
Insulin-like growth factor binding protein (IGFBP)-5 is a conserved protein synthesized and secreted by vascular smooth muscle cells (VSMCs). IGFBP-5 binds to extracellular IGFs and modulates IGF actions in regulating VSMC proliferation, migration, and survival. IGFBP-5 also stimulates VSMC migration through an IGF-independent mechanism, but the molecular basis underlying this ligand-independent action is unknown. In this study, we show that endogenous IGFBP-5 or transiently expressed IGFBP-5-EGFP, but not IGFBP-4-EGFP, is localized in the nuclei of VSMCs. Using a series of IGFBP-4/5 chimeras and IGFBP-5 points mutants, we demonstrated that the IGFBP-5 C-domain is necessary and sufficient for its nuclear localization, and residues K206, K208, K217, and K218 are particularly critical. Intriguingly, inhibition of protein secretion abolishes IGFBP-5 nuclear localization, suggesting the nuclear IGFBP-5 is derived from the secreted protein. When added exogenously, (125)I- or Cy3-labeled IGFBP-5 is capable of cellular entry and nuclear translocation. To identify potential transcriptional factor(s) that interact with IGFBP-5, a human aorta cDNA library was screened by a yeast two-hybrid screening strategy. Although this screen identified many extracellular and cytosolic proteins that are known to interact with IGFBP-5, no known transcription factors were found. Further motif analysis revealed that the IGFBP-5 N-domain contains a putative transactivation domain. When fused to GAL-4 DNA dinging domain and tested, the IGFBP-5 N-domain has strong transactivation activity. Mutation of the IGF binding domain or treatment of cells with IGF-I has little effect on transactivation activity. These results suggest that IGFBP-5 is localized in VSMC nucleus and possesses transcription-regulatory activity that is IGF independent.
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor 48109-1048, USA.
Qijin
Q
Xu
Shenghua
S
Li
Yang
Y
Zhao
Travis J
TJ
Maures
Ping
P
Yin
Cunming
C
Duan
D020816:Amino Acid Motifs; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry*; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000235:genetics; D004705:Endocytosis; D019143:Evolution, Molecular; D006801:Humans; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000737:chemistry / Q000235:genetics; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism* / Q000648:ultrastructure; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000494:pharmacology / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013552:Swine; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology; D014162:Transfection; D015027:Zebrafish / Q000235:genetics
D018076:DNA, Complementary; D018974:Insulin-Like Growth Factor Binding Protein 4; D018975:Insulin-Like Growth Factor Binding Protein 5; D011993:Recombinant Fusion Proteins