Identification of four alternatively spliced transcripts of the Ucma/GRP gene, encoding a new Gla-containing protein

Exp Cell Res. 2010 Jan 15;316(2):203-15. doi: 10.1016/j.yexcr.2009.10.002. Epub 2009 Oct 9.

Abstract

The Ucma protein (Upper zone of growth plate and cartilage matrix associated protein) has recently been described as a novel secretory protein mainly expressed in cartilage and also as a novel vitamin-K-dependent protein named GRP (Gla-rich protein). This protein has the highest Gla content of any protein known to date. In this article, we identify four alternatively spliced variants of Ucma/GRP gene transcripts in mouse chondrocytes. We show that the expression of all four isoforms is associated with the early stages of chondrogenesis. The Ucma/GRP gene encodes four proteins named Ucma/GRP-F1, -F2, -F3, and -F4, which differ by exon 2, exon 4, or both. Among them, only Ucma/GRP-F1 and -F3 were secreted into the culture medium of transfected chondrocytes, while Ucma/GRP-F2 and -F4 accumulated in the cells. Using HeLa cells or freshly isolated embryonic mouse chondrocytes transfected with enhanced green fluorescent protein fusions, microscopy analysis revealed that Ucma/GRP-F1 and -F3 were localized in the Golgi complex, whereas Ucma/GRP-F2 and -F4 formed aggregates. This aggregation was microtubule-dependent since disruption of microtubules with nocodazole reduced Ucma/GRP-F2 and -F4 aggregation in a reversible manner. Using biochemical fractionation and Western blot analysis, Ucma/GRP-F1 and -F3 isoforms were detected in the soluble fraction while Ucma/GRP-F2 and -F4 were found in an insoluble-enriched fraction. We conclude that the co-expression of soluble and insoluble isoforms also Gla-rich and Gla-deleted isoforms may be finely tuned. Imbalance in isoform expression may therefore be involved in skeletal pathology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Carboxyglutamic Acid / analysis*
  • Alternative Splicing / genetics*
  • Animals
  • Bone Morphogenetic Protein 2 / pharmacology
  • Cell Differentiation / physiology
  • Chondrocytes / metabolism
  • Chondrogenesis / physiology
  • Cytoplasm / metabolism
  • Embryo, Mammalian / cytology
  • Embryo, Mammalian / metabolism
  • Exons / genetics
  • Extracellular Matrix Proteins
  • Gene Expression / drug effects
  • Gene Expression / genetics
  • Gene Expression Regulation, Developmental / physiology
  • Golgi Apparatus / metabolism
  • HeLa Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • Mice
  • Mice, Transgenic
  • Microtubules / drug effects
  • Microtubules / metabolism
  • Molecular Sequence Data
  • Nocodazole / pharmacology
  • Organelles / metabolism
  • Protein Isoforms / chemistry
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • Proteins / chemistry
  • Proteins / genetics*
  • Proto-Oncogene Protein c-fli-1 / genetics
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Transforming Growth Factor beta1 / pharmacology

Substances

  • Bone Morphogenetic Protein 2
  • Extracellular Matrix Proteins
  • Fli1 protein, mouse
  • Intracellular Signaling Peptides and Proteins
  • Protein Isoforms
  • Proteins
  • Proto-Oncogene Protein c-fli-1
  • Recombinant Fusion Proteins
  • Transforming Growth Factor beta1
  • Ucma protein, mouse
  • 1-Carboxyglutamic Acid
  • Nocodazole

Associated data

  • GENBANK/FJ217397
  • GENBANK/FJ217398
  • GENBANK/FJ217399
  • GENBANK/FJ217400