Regulated expression of active biotinylated G-protein coupled receptors in mammalian cells.

We have developed a mammalian expression system suitable for the production of enzymatically biotinylated integral membrane proteins. The key feature of this system is the doxycycline (dox)-regulated co-expression of a secreted variant of Escherichia coli biotin ligase (BirA) and a target protein with a 13-residue biotin acceptor peptide (BioTag) appended to its extracellular domain. Here we describe the expression and functional analysis of three G-protein coupled receptors (GPCRs): protease-activated receptors (PARs) 1 and 2, and the platelet ADP receptor, P2Y(12). Clonal Chinese hamster ovary (CHO) Tet-On cell lines that express biotinylated GPCRs were rapidly isolated by fluorescence-activated cell sorting following streptavidin-FITC staining, thereby circumventing the need for manual colony picking. Analysis by Western blotting with streptavidin-HRP following endoglycosidase treatment revealed that all three GPCRs undergo N-linked glycosylation. The expression of biotinylated GPCRs on the cell surface was regulated by the concentration of dox in the medium, reaching a maximum at approximately 1 microg/mL dox. Similarly, the extent of GPCR biotinylation was dependent on biotin concentration, with maximum and complete biotinylation achieved upon supplementation with 50 microM biotin. Biotinylated PAR1 and PAR2 were readily and specifically cleaved on the surface of intact cells by their cognate proteases, and were capable of transducing extracellular stimuli, resulting in the downstream phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Notably, P2Y(12) mediated agonist-induced ERK phosphorylation only when it was expressed at low levels on the cell surface, highlighting the utility of regulated expression for the production of functionally active GPCRs in mammalian cells.

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

Proline-rich Gla protein 2 (PRGP2) is one of four known vertebrate transmembrane gamma-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 gamma-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 gamma-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.

A versatile system for site-specific enzymatic biotinylation and regulated expression of proteins in cultured mammalian cells.

We have developed a system for producing biotinylated recombinant proteins in mammalian cells. The expression construct consists of an inducible tetracycline response element (TRE) that drives expression of a bicistronic cassette comprising a biotin acceptor peptide (BioTag) fused to either terminus of the target protein, the gene for Escherichia coli biotin ligase (BirA), and an intervening internal ribosome entry site (IRES). By either transient or stable transfection of Chinese hamster ovary (CHO) Tet-On cells, we successfully expressed, detected, and immobilized biotinylated human Itch, a pleiotropic multi-domain ubiquitin-protein ligase, as well as Gla-RTK, a putative vitamin K-dependent receptor tyrosine kinase. The biotinylation of recombinant Itch in transiently transfected CHO Tet-On cells required biotin supplementation and coexpression of BirA, occurred quantitatively and specifically on the lysine residue of the BioTag, and enabled detection of Itch by Western blot in as little as 10ng of total lysate protein. Stably selected clones were rapidly pre-screened for doxycycline (dox)-inducible BirA expression by ELISA, and subsequently screened for dox-inducible expression of biotinylated Itch. Biotinylated Gla-RTK was detectable in as little as 5ng of total lysate protein from transiently transfected CHO Tet-On cells, and exhibited pronounced tyrosine phosphorylation. In stable clones however, constitutive phosphorylation was prevented by reducing the expression level of Gla-RTK through the titration of dox. These results demonstrate the utility of this system for the expression of 'difficult' proteins, particularly those that are cytotoxic or those that may require lower expression levels to ensure appropriate post-translational modification.

Vitamin K-dependent proteins in Ciona intestinalis, a basal chordate lacking a blood coagulation cascade.

We have isolated and sequenced several cDNAs derived from the sea squirt Ciona intestinalis that encode vitamin K-dependent proteins. Four of these encode gamma-carboxyglutamic acid (Gla) domain-containing proteins, which we have named Ci-Gla1 through Ci-Gla4. Two additional cDNAs encode the apparent orthologs of gamma-glutamyl carboxylase and vitamin K epoxide reductase. Ci-Gla1 undergoes gamma-glutamyl carboxylation when expressed in CHO cells and is homologous to Gla-RTK, a putative receptor tyrosine kinase previously identified in a related ascidian. The remaining three Gla domain proteins are similar to proteins that participate in fundamental developmental processes, complement regulation, and blood coagulation. These proteins are generally expressed at low levels throughout development and exhibit either relatively constant expression (Ci-Gla1, gamma-glutamyl carboxylase, and vitamin K epoxide reductase) or spatiotemporal regulation (Ci-Gla2, -3, and -4). These results demonstrate the evolutionary emergence of the vitamin K-dependent Gla domain before the divergence of vertebrates and urochordates and suggest novel functions for Gla domain proteins distinct from their roles in vertebrate hemostasis. In addition, these findings highlight the usefulness of C. intestinalis as a model organism for investigating vitamin K-dependent physiological phenomena, which may be conserved among the chordate subphyla.