Abstract
The hyaluronan receptor for endocytosis (HARE), or Stabilin-2, is the mammalian endocytic clearance receptor for HA, heparin, advanced glycation end-products, acetylated and oxidized low-density lipoproteins and collagen N-terminal propeptides. This large 2551 amino acid receptor is encoded by a gene that covers over 180 kbp on human chromosome 12 and is predicted to be composed of 69 exons. Due to the expression profile of this gene and the number of exons it contains, we hypothesized that splice variants of stab2 are encoded in these tissues. In addition, a correlation between alternative splice variants and cancer progression has been shown in other HA receptors such as RHAMM and CD42. In this study, two methods were utilized in identifying and/or isolating the HARE splice variants. The first method used primer sets to amplify the 190-HARE encoding region that could contain splice junctions; therefore, they were purified from agarose gels and sequenced. Five splice variants were detected in that manner. In the second approach, the entire open reading frame of HARE was amplified. This allowed four splice variants with extensive exon splicing to be isolated. After the splice variants were sequenced, three were cloned into a mammalian expression vector. Next, stable cell lines expressing the variants were created in order to determine stable protein expression. In this study, the splice variants were found to be tissue specific in most cases. This suggests that tissue specific regulatory splicing mechanisms may lead to differences in functionality between the splice variants.