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

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.