Membrane cholesterol modulates STEAP2 conformation during dynamic intracellular trafficking processes leading to broad subcellular distribution
Introduction
STEAP2 belongs to the Six-Transmembrane Epithelial Antigen of the Prostate (STEAP) protein family and was originally identified as a protein overexpressed in prostate cancer [1], [2]. Because its expression is widely elevated in different cancers, STEAP2 is now considered as an important diagnostic maker and therapeutic target [2], [3], [4]. After the demonstration of STEAP3's function as a metalloreductase and its physiological link to transferrin-mediated iron uptake in erythroid cells using mouse models [5], both STEAP2 and STEAP4 were biochemically shown to possess metalloreductase activity in vitro [6], [7]. The metalloreductase function of STEAP1 is somewhat controversial because it has a shorter N-terminal cytosolic region devoid of oxidoreductase domain [6] although it retains a conserved FAD binding site and metal binding motif [8]. However, in a recent study, a detergent-solubilized recombinant STEAP1 was shown to possess both metalloreductase and superoxide synthase activities [9]. Those studies conducted over a decade period collectively support that STEAP family members are metalloreductases and they function in different cell types and in different subcellular locations [3].
Although biochemical enzymatic activity of STEAP2 appears evident [6], [7], the precise subcellular localization and the intracellular site of STEAP2 function remain controversial. For example, a GFP-tagged STEAP2 construct not only localized to the plasma membrane but also at various cytoplasmic vesicular structures of unknown identity in DU145 cells [1]. Other investigators, using COS-1 cells, found that STEAP2 co-localized with the markers of trans-Golgi network and early endosomes, and to a lesser extent to the plasma membrane, which led them to propose that STEAP2 is associated with membrane trafficking functions of both secretory and endocytic pathways [2]. Yet another group showed evidence of complete co-localization of STEAP2 with transferrin and the transferrin receptor in the endosomes of HEK293T cells and concluded that STEAP2 is an endosomal metalloreductase [6]. Similar inconsistency and discrepancy on the subcellular localizations are also apparent in STEAP3 and STEAP4 by comparing published results from different groups [5], [6], [7], [8], [10], [11], [12], [13].
A cis-acting YxxΦ motif located in the first cytosolic loop has been proposed to function as an endosome targeting motif in STEAP3 [11]. Although this motif is conserved in other STEAP members, the fact that different STEAP members show different steady state subcellular localization suggests that the function of this motif is not equal in different STEAP members. It is currently unknown whether there are additional cis-acting elements that are responsible for STEAP2's broad and seemingly inconsistent subcellular distribution in different cell types. In addition, trans-acting factors that might influence the localization, structure or function of STEAP2 have not been described to date. Whether there are any biophysical differences in STEAP2 when it localizes to different membrane compartments are also unknown. We postulate that presence of such difference could potentially provide a mechanism to regulate the properties of STEAP2 in a compartment-specific fashion despite broad cellular distribution.
During a course of antibody discovery program, we identified a unique monoclonal antibody (mAb) (clone 1D11) that recognizes an epitope located in the second extracellular loop of human STEAP2. Using the 1D11 mAb, along with other detection reagents in combination with various imaging techniques, we thoroughly characterized the subcellular localization of STEAP2 in transfected HEK293 cells and prostate cancer cell lines expressing endogenous STEAP2. Although the majority of STEAP2 were present in the juxtanuclear Golgi region and endosome-like puncta at steady state, the 1D11 mAb allowed us to selectively visualize and characterize the cell surface localizing pool of STEAP2 and the fate of STEAP2 after reaching the cell surface. Conformation-sensitive nature of the 1D11 epitope additionally helped us uncover a novel conformational modulation of STEAP2 dependent upon the local membrane cholesterol level. Despite the confirmed transient cell surface localization, STEAP2's metalloreductase activity appeared to be tightly suppressed at the cell surface in a live cell assay that can readily detect the cell surface metalloreductase activity of STEAP4. Taken together, the results raise a possibility that STEAP2's metalloreductase activity is regulated in a conformation- and localization-dependent fashion even when STEAP2 is broadly and dynamically distributed to various membrane compartments at steady state. A key role of pre-existent cholesterol content difference among different cellular membranes is suggested in the STEAP2's conformational and functional modulation.
Section snippets
STEAP2 broadly distributes to the Golgi, cytoplasmic puncta, and plasma membrane
Studies from various laboratories showed that STEAP2 resides in multiple membrane compartments at different ratio [1], [2], [6], but how such broad subcellular distribution is maintained remains unclear. To address the complex nature of STEAP2 subcellular distribution, we first made a C-terminally FLAG-tagged full-length STEAP2 construct (Fig. 1A, second row; Fig. 1F, lane 1) and examined its localization by indirect immunofluorescent microscopy using transfected HEK293 cells. To detect the
Underlying reasons for broad subcellular distribution of STEAP2
Unlike typical organelle resident proteins that are targeted to specific membrane compartments, STEAP2 is broadly distributed into multiple subcellular compartments including the plasma membrane [1], [2], [6]. As a result, it has been unclear as to which compartment is its functional destination and where the proposed metalloreductase activity actually takes place. This study demonstrated that a broad subcellular distribution is maintained by a fine balance between the secretory trafficking of
Detection antibodies
Rabbit polyclonal anti-STEAP2 (Millipore, cat. ABC341) was used for immunofluorescent imaging. Rabbit polyclonal anti-STEAP2 antibodies used for Western blotting were either from ProteinTech Group Inc. (cat. 20201-1-AP) or Thermo Fisher Scientific (PA5-33062). Two additional rabbit polysera against STEAP2 were evaluated and they were from Thermo Fisher Scientific (cat. PA5-20405 and PA5-25494). A hybridoma cell line producing the mouse monoclonal antibody against human STEAP1 (clone 120.545)
Acknowledgement
The authors thank colleagues in Amgen Protein Technology group to generate various reagents used for mouse immunization and Amgen Biologic Discovery group for generating mouse anti-human STEAP2 monoclonal antibodies from hybridoma campaigns.
Author contribution
Study conception and design: HH
Key reagent generation: BA, DP, ZX, MRD, PL, JZ, JZ, DL, CM
Acquisition of data: HH, CL, CM
Analysis and interpretation of data: HH, CL, CM
Drafting of manuscript: HH
Critical revision: HH, CL, CM
Final approval of manuscript: HH, CL, BA, DP, ZX, MRD, PL, JZ, JZ, DL, CM, ACL
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