Novel protein interactors of urokinase-type plasminogen activator receptor

Abstract

The urokinase-type plasminogen activator receptor (uPAR) has been implicated in tumor growth and metastasis. The crystal structure of uPAR revealed that the external surface is largely free to interact with a number of proteins. Additionally, due to absence of an intracellular cytoplasmic protein domain, many of the biological functions of uPAR necessitate interactions with other proteins. Here, we used yeast two-hybrid screening of breast cancer cDNA library to identify hSpry1 and HAX1 proteins as putative candidate proteins that interact with uPAR bait constructs. Interaction between these two candidates and uPAR was confirmed by GST-pull down, co-immunoprecipitation assays and confocal microscopy. These novel interactions that have been identified may also provide further evidence that uPAR can interact with a number of other proteins which may influence a range of biological functions.

Introduction

The urokinase plasminogen activator receptor (uPAR) has a well described role in extracellular matrix (ECM) proteolysis, and other important functions in cell growth, adhesion, migration and invasion [1]. These latter additional roles are influenced by the ability of uPAR to engage in multiple protein–protein interactions [2]. In addition to the primary ligand uPA that binds to uPAR, there are two further ligands, which have been found to bind uPAR. These are vitronectin (VN) [3], and kininogen [4]. Furthermore, uPAR is linked to the cell membrane via a GPI anchor, and so has no intracellular domain [5]. Hence, many of the biological functions of uPAR necessitate interactions with other proteins on the cell surface, in particular, transmembrane proteins. The high lateral mobility of uPAR on the cell membrane may provide the mechanism by which it associates with other transmembrane receptors [6]. Indeed, the recent crystal structure of the soluble form of uPAR was solved at 2.7 Å in association with a competitive peptide inhibitor of the uPA–uPAR interaction [7]. The proposed model for the uPA/uPAR interaction revealed uPA bound to uPAR via a central cavity. This model left the external receptor surface free to bind and interact with other proteins [7].

In addition to the primary ligand uPA, a number of uPAR specific interactions have also been identified and are consistent with the varied roles regulated by uPAR including cell adhesion, cell migration, angiogenesis, apoptosis and cancer metastasis. Indeed, there is now evidence that uPAR can bind with the cell surface β₁, β₂, β_3, β₅ and β₆ integrin subfamilies members [8], [9], [10], [11]. Other associations and physical binding partners include chemotactic receptors [12], the epidermal growth factor receptor (EGFR) [13], α2-macroglobulin receptor/low density lipoprotein receptor-related protein [14], GP130 [15], the mannose-6-phosphate/insulin-like growth factor 2 receptor [16], and Mrj [17]. However, it is believed that there should be other unidentified proteins that interact with uPAR.

The amino acid sequence of uPAR has three repeats, approximately 90 amino acids each, suggesting the existence of three homologous independently folded domains. These have been designated DI, DII and DIII from the N-terminal end [18]. These three domains are homologous to the Ly6/neurotoxin family and have two short linker regions between DI and DII, as well as, DII and DIII. Loop 3 of DI and in particular, the amino acid Arg⁵³, Leu⁵⁵, Tyr⁵⁷ and Leu⁶⁶ are important in the ligand binding of uPA [19], [20]. Chymotrypsin can cleave uPAR in the linker region between DI and DII at residue 87 and uPA similarly cleaves at residue 84 to result in a cleaved form of uPAR [21]. There is also soluble uPAR (suPAR) that may be either the full-length receptor or a truncated form consisting of only DII and DIII [22]. In the present work, we have used yeast two-hybrid (Y2H) to identify novel interacting proteins by utilizing a number of alternate bait constructs based on uPAR. The screening of a breast cancer cDNA library identified hSpry1 and HAX1 proteins as novel candidate proteins that interact with uPAR. These novel protein–protein interactions were confirmed by GST-pull down, co-immunoprecipitation assays and confocal microscopy.