The non-collagenous C-terminal domain of the ␣ 3 chain of collagen IV is the autoantigen in Goodpasture disease, an autoimmune disorder described only in humans. Specific N-terminal phosphorylation is a biological feature unique to the human domain when compared with other homologous domains lacking immunopathogenic potential. We have recently cloned from a HeLa-derived cDNA library a novel serine/threonine kinase (Goodpasture antigen-binding protein (GPBP)) that phosphorylates the N-terminal region of the human domain (Raya, A. Revert, F, Navarro, S. and Saus J. (1999) J. Biol. Chem. 274, 12642-12649). We show here that the pre-mRNA of GPBP is alternatively spliced in human tissues and that the most common transcript found encodes GPBP⌬26, a molecular isoform devoid of a 26-residue serine-rich motif. Recombinantly expressed GPBP⌬26 exhibits lower activity than GPBP, due at least in part to a reduced ability of GPBP⌬26 to interact and to form very active high molecular weight aggregates. In human tissues, GPBP shows a more limited expression than GPBP⌬26 but displays a remarkable preference for the small vessels and for histological structures targeted by natural autoimmune responses including alveolar and glomerular basement membranes, the two main targets in Goodpasture disease. GPBP expression is, in turn, up-regulated in the striated muscle of a Goodpasture patient and in other autoimmune conditions including cutaneous lupus erythematosus, pemphigoid, and lichen planus.

Goodpasture-antigen binding protein (GPBP) is a nonconventional Ser/Thr kinase for basement membrane type IV collagen.Various studies have questioned these findings and proposed that GPBP serves as transporter of ceramide between the endoplasmic reticulum and the Golgi apparatus. Here we show that cells expressed at least two GPBP isoforms resulting from canonical (77-kDa) and noncanonical (91-kDa) mRNA translation initiation. The 77-kDa polypeptide interacted with type IV collagen and localized as a soluble form in the extracellular compartment. The 91-kDa polypeptide and its derived 120-kDa polypeptide associated with cellular membranes and regulated the extracellular levels of the 77-kDa polypeptide. A short motif containing two phenylalanines in an acidic tract and the 26-residue Ser-rich region were required for efficient 77-kDa polypeptide secretion. Removal of the 26-residue Ser-rich region by alternative exon splicing rendered the protein cytosolic and sensitive to the reduction of sphingomyelin cellular levels. These and previous data implicate GPBPs in a multicompartmental program for protein secretion (i.e. type IV collagen) that includes: 1) phosphorylation and regulation of protein molecular/supramolecular organization and 2) interorganelle ceramide trafficking and regulation of protein cargo transport to the plasma membrane. Goodpasture antigen-binding protein (GPBP)2 phosphorylates the noncollagenous-1 (NC1) domain of the ␣3 chain of type IV collagen (␣3(IV)NC1) (1). This domain is a pivotal structure in the molecular and supramolecular organization of the glomerular basement membrane collagen and also the target of autoantibodies mediating glomerulonephritis in Goodpasture disease (2). Increased GPBP expression has been associated with autoimmune pathogenesis including Goodpasture disease (3) and with the induction of glomerular basement membrane collagen disorganization and deposit of IgA antibodies (4). These observations suggest that GPBP regulates glomerular basement membrane collagen organization and induces type IV collagen-based antibody-mediated glomerulonephritis when its expression is abnormally elevated (3, 4). The GPBP gene (COL4A3BP) also encodes for GPBP⌬26, a more abundant, less active, alternatively spliced GPBP variant lacking a 26-residue Ser-rich region that is apparently not regulated under these pathological conditions (3).GPBP contains multiple structural elements including N-terminal pleckstrin homology domain, Ser-Xaa-Yaa region, bipartite nuclear localization signal, coiled-coil domain, two phenylalanines in an acidic tract (FFAT) motif, and C-terminal steroidogenic acute regulatory related lipid transfer (START) domain. Additional structural features include motifs for selfinteraction and phosphorylation (1, 3, 5, 6). The pleckstrin homology domains comprise a variety of poorly conserved structures present only in eukaryotes that have been proposed to mediate protein targeting to cellular membranes through interaction with phosphoinositides (7). A variety of proteins inc...

Increased expression of Goodpasture antigen-binding protein (GPBP), a protein that binds and phosphorylates basement membrane collagen, has been associated with immune complex-mediated pathogenesis. However, recent reports have questioned this biological function and proposed that GPBP serves as a cytosolic ceramide transporter (CERT L ). Thus, the role of GPBP in vivo remains unknown. New Zealand White (NZW) mice are considered healthy animals although they convey a genetic predisposition for immune complex-mediated glomerulonephritis. Here we show that NZW mice developed age-dependent lupus-prone autoimmune response and immune complex-mediated glomerulonephritis characterized by elevated GPBP, glomerular basement membrane (GBM) collagen disorganization and expansion, and deposits of IgA on disrupted GBM. Transgenic overexpression of human GPBP (hGPBP) in non-lupusprone mice triggered similar glomerular abnormalities including deposits of IgA on a capillary GBM that underwent dissociation, in the absence of an evident autoimmune response. We provide in vivo evidence that GPBP regulates GBM collagen organization and its elevated expression causes dissociation and subsequent accumulation of IgA on the GBM. Finally, we describe a previously unrecognized pathogenic mechanism that may be relevant in human primary immune complex-mediated glomerulonephritis. (Am J Pathol

Goodpasture antigen-binding protein (GPBP) is an exportable1 Ser/Thr kinase that induces collagen IV expansion and has been associated with chemoresistance following epithelial-to-mesenchymal transition (EMT). Here we demonstrate that cancer EMT phenotypes secrete GPBP (mesenchymal GPBP) which displays a predominant multimeric oligomerization and directs the formation of previously unrecognized mesh collagen IV networks (mesenchymal collagen IV). Yeast twohybrid (YTH) system was used to identify a 260 SHCIE 264 motif critical for multimeric GPBP assembly which then facilitated design of a series of potential peptidomimetics. The compound 3-[4''-methoxy-3,2'-dimethyl-(1,1';4',1'')terphenyl-2''-yl]propionic acid, or T12, specifically targets mesenchymal GPBP and disturbs its multimerization without affecting kinase catalytic site. Importantly, T12 reduces growth and metastases of tumors populated by EMT phenotypes. Moreover, low-dose doxorubicin sensitizes epithelial cancer precursor cells to T12, thereby further reducing tumor load. Given that T12 targets the pathogenic mesenchymal GPBP, it does not bind significantly to normal tissues and therapeutic dosing was not associated with toxicity. T12 is a first-in-class drug candidate to treat cancer by selectively targeting the collagen IV of the tumor cell microenvironment. www.impactjournals.com/oncotarget/

The Goodpasture antigen‐binding protein, GPBP, is a serine/threonine kinase whose relative expression increases in autoimmune processes. Tumor necrosis factor (TNF) is a pro‐inflammatory cytokine implicated in autoimmune pathogenesis. Here we show that COL4A3BP, the gene encoding GPBP, maps head‐to‐head with POLK, the gene encoding for DNA polymerase kappa (pol κ), and shares with it a 140‐bp promoter containing a Sp1 site, a TATA‐like element, and a nuclear factor kappa B (NFκB)‐like site. These three elements cooperate in the assembly of a bidirectional transcription complex containing abundant Sp1 and little NFκB that is more efficient in the POLK direction. Tumour necrosis factor cell induction is associated with Sp1 release, NFκB recruitment and assembly of a complex comparatively more efficient in the COL4A3BP direction. This is accomplished by competitive binding of Sp1 and NFκB to a DNA element encompassing a NFκB‐like site that is pivotal for the 140‐bp promoter to function. Consistently, a murine homologous DNA region, which contains the Sp1 site and the TATA‐like element but is devoid of the NFκB‐like site, does not show transcriptional activity in transient gene expression assays. Our findings identify a human‐specific TNF‐responsive transcriptional unit that locates GPBP in the signalling cascade of TNF and substantiates previous observations, which independently related TNF and GPBP with human autoimmunity.

Background: GPBP-1 is a non-conventional kinase that regulates glomerular basement membrane collagen organization. Results: GPBP-1 targets GIP130, a new myosin-binding protein, and regulates myofibrillogenesis in cultured myoblasts. Conclusion: GPBP-1 regulates myofibril formation. Significance: GPBP-1 is a kinase for structural protein organization at both intracellular and extracellular compartments.

The noncollagenous-1 domain of the alpha3 chain of collagen IV networks of basement membranes is the target of an antibody-mediated inflammatory response in Goodpasture autoimmune disease. This domain when excised from basement membranes by bacterial collagenase digestion exists in two molecular forms, M(H) and M(L), that differ in cleavage site and mobility in SDS-PAGE. In the present study, M(H) and M(L) were shown to also differ with respect to epitope exposure, susceptibility to endoprotease digestion, and redox states of specific cystene residues, as determined by MS. Moreover, M(H) and M(L) assemble to form different quaternary structures, critically influencing pathogenic epitope(s) exposure and autoantibody binding. Collectively, our findings reveal that M(H) and M(L) are conformational isomers stabilized by a distinct disulfide bond connectivity, and coexist in basement membranes. The hitherto unrecognized conformational diversification of the Goodpasture autoantigen may be of relevance in pathogenesis.

The emergence of the basement membrane (BM), a specialized form of extracellular matrix, was essential in the unicellular transition to multicellularity. However, the mechanism is unknown. Goodpasture antigen-binding protein (GPBP), a BM protein, was uniquely poised to play diverse roles in this transition owing to its multiple isoforms (GPBP-1,-2, and-3) with varied intracellular and extracellular functions (ceramide trafficker and protein kinase). We sought to determine the evolutionary origin of GPBP isoforms. Our findings reveal the presence of GPBP in unicellular protists, with GPBP-2 as the most ancient isoform. In vertebrates, GPBP-1 assumed extracellular function that is further enhanced by membrane-bound GPBP-3 in mammalians, whereas GPBP-2 retained intracellular function. Moreover, GPBP-2 possesses a dual intracellular/extracellular function in cnidarians, an early nonbilaterian group. We conclude that GPBP functioning both inside and outside the cell was of fundamental importance for the evolutionary transition to animal multicellularity and tissue evolution.