Proline-rich Gla protein 2 is a cell-surface vitamin K-dependent protein that binds to the transcriptional coactivator Yes-associated protein

Abstract

Proline-rich Gla protein 2 (PRGP2) is one of four known vertebrate transmembrane ␥-carboxyglutamic acid (Gla) proteins. Members of this protein family are broadly expressed in fetal and adult human tissues and share a common architecture consisting of a predicted propeptide and Gla domain, a single-pass transmembrane segment, and tandem Pro/Leu-Pro-Xaa-Tyr (PY) motifs near their C termini. Using a methodology developed for the regulated expression of enzymatically biotinylated proteins in mammalian cells, we demonstrate that PRGP2 undergoes ␥-glutamyl carboxylation in a manner that is both dependent upon the presence of a proteolytically cleavable propeptide and sensitive to warfarin, a vitamin K antagonist that is widely used as an antithrombotic agent. When expressed at physiologically relevant levels, the majority of PRGP2 is present in the ␥-glutamyl carboxylated, propeptide-cleaved (mature) form. We additionally demonstrate, by Western blotting and flow cytometry, that mature PRGP2 is predominantly located on the cell surface with the Gla domain exposed extracellularly. In a yeast two-hybrid screen that used the C-terminal cytoplasmic region of PRGP2 as bait, we identified the WW domain-containing transcriptional coactivator Yes-associated protein (YAP) as a binding partner for PRGP2. In GST pull-down experiments, both PRGP2 PY motifs and both YAP WW domains were essential for complex formation, as were residues proximal to the core sequence of the first PY motif. These findings suggest that PRGP2 may be involved in a signal transduction pathway, the impairment of which may be an unintended consequence of warfarin therapy.Gla domain ͉ warfarin ͉ WW domain

Cellular Localization and Characterization of Cytosolic Binding Partners for Gla Domain-containing Proteins PRRG4 and PRRG2

“…2 B ). These results are consistent with a previous study reporting that the related protein PRRG2 is localized to the plasma membrane (16). …”

“…The only proteins that are known to interact with PRRG proteins are YAP1 and NEDD4, both of which can interact with PRRG2 (15, 16). NEDD4 is a ubiquitin ligase, and YAP1 is a downstream target of the Hippo tumor suppressor pathway (17).…”

Mammalian Alpha Arrestins Link Activated Seven Transmembrane Receptors to Nedd4 Family E3 Ubiquitin Ligases and Interact with Beta Arrestins

“…The many published successes with PY-WW yeast two hybrid interactions show that neither PY motifs nor WW domains are generally prone to self-activating false positives. [40] There are two classes of aArr PY motifs defined by their positions, distance apart and conserved flanking sequence (ARRDC1 and TXNIP/ARRDC2/3/4). [15] We tested eight combinations of tail fragments (containing both PY motifs) from ARRDC1 and 3 with complete WW regions of select candidate partners.…”

Structural insights into the functional versatility of WW domain-containing oxidoreductase tumor suppressor

“…In addition to WBP1/2 (23, 24), ErbB4 (25, 26) and p73 (27), YAP is also known to interact with a wide variety of other cellular proteins such as TMG2 transmembrane protein (28), PTPN14 phosphatase (29), SMAD7 signaling adaptor (30) and PTCH1 transmembrane receptor (31). Importantly, it is believed that WWOX antagonizes the transactivation function of YAP by virtue of its ability to competitively bind to these proteins and, in so doing, plays a central role in the maintenance of cellular homeostasis (15, 19).…”