Benedikte Jacobsen

Structure and ligand interactions of the urokinase receptor (uPAR).

The urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycolipid-anchored membrane glycoprotein, which is responsible for focalizing plasminogen activation to the cell surface through its high-affinity binding to the serine protease uPA. This tight interaction (KD less than 1 nM) is accomplished by an unusually large and hydrophobic binding cavity in uPAR that is created by a unique interdomain assembly involving all three homologous domains of the receptor. These domains belong to the Ly-6/uPAR (LU) protein domain family, which is defined by a consensus sequence predominantly based on disulfide connectivities, and they adopt a characteristic three-finger fold. Interestingly, the gene for uPAR is localized in a cluster of 6 homologous genes encoding proteins with multiple LU-domains. The structural biology of uPAR will be reviewed with special emphasis on its multidomain composition and the interaction with its natural protein ligands, i.e. the serine protease uPA and the matrix protein vitronectin.

Rational Targeting of the Urokinase Receptor (uPAR): Development of Antagonists and Non-Invasive Imaging Probes

In the last two decades, the urokinase-type plasminogen activator receptor (uPAR) has been implicated in a number of human pathologies such as cancer, bacterial infections, and paroxysmal nocturnal hemoglobinuria. The primary function of this glycolipid-anchored receptor is to focalize uPA-mediated plasminogen activation at the cell surface, which is accomplished by its high-affinity interaction with the growth factor-like domain of uPA. Detailed insights into the molecular basis underlying the interactions between uPAR and its two bona fide ligands, uPA and vitronectin, have been obtained recently by X-ray crystallography and surface plasmon resonance studies. Importantly, these structural studies also define possible druggable target sites in uPAR for small molecules and provide guidelines for the development of reporter groups applicable for non-invasive molecular imaging of uPAR expression in vivo by positron emission tomography. In this review, we will discuss recent advances in our perception of the structure-function relationships of uPAR ligation and how these may assist translational research in preclinical intervention studies of uPAR function.

Conformational Regulation of Urokinase Receptor Function IMPACT OF RECEPTOR OCCUPANCY AND EPITOPE-MAPPED MONOCLONAL ANTIBODIES ON LAMELLIPODIA INDUCTION

The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein with an established role in focalizing uPA-mediated plasminogen activation on cell surfaces. Distinct from this function, uPAR also modulates cell adhesion and migration on vitronectin-rich matrices. Although uPA and vitronectin engage structurally distinct binding sites on uPAR, they nonetheless cooperate functionally, as uPA binding potentiates uPAR-dependent induction of lamellipodia on vitronectin matrices. We now present data advancing the possibility that it is the burial of the β-hairpin in uPA per se into the hydrophobic ligand binding cavity of uPAR that modulates the function of this receptor. Based on these data, we now propose a model in which the inherent interdomain mobility in uPAR plays a major role in modulating its function. Particularly one uPAR conformation, which is stabilized by engagement of the β-hairpin in uPA, favors the proper assembly of an active, compact receptor structure that stimulates lamellipodia induction on vitronectin. This molecular model has wide implications for drug development targeting uPAR function.

Mimicry of the Regulatory Role of Urokinase in Lamellipodia Formation by Introduction of a Non-native Interdomain Disulfide Bond in Its Receptor

Background: The urokinase receptor (uPAR) acts as a modulator of lamellipodia formation on vitronectin-rich matrices. Results: Constraining the flexibility of uPAR by an interdomain cross-link drives it into a constitutively active state. Conclusion: Conformational dynamics of uPAR is important for its function and is regulated by uPA binding. Significance: This flexibility needs to be considered when investigating and targeting the function of uPAR. The high-affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays a regulatory role for both extravascular fibrinolysis and uPAR-mediated adhesion and migration on vitronectin-coated surfaces. We have recently proposed that the adhesive function of uPAR is allosterically regulated via a “tightening” of its three-domain structure elicited by uPA binding. To challenge this proposition, we redesigned the uPAR structure to limit its inherent conformational flexibility by covalently tethering domains DI and DIII via a non-natural interdomain disulfide bond (uPARH47C-N259C). The corresponding soluble receptor has 1) a smaller hydrodynamic volume, 2) a higher content of secondary structure, and 3) unaltered binding kinetics towards uPA. Most importantly, the purified uPARH47C-N259C also displays a gain in affinity for the somatomedin B domain of vitronectin compared with uPARwt, thus recapitulating the improved affinity that accompanies uPA-uPARwt complex formation. This functional mimicry is, intriguingly, operational also in a cellular setting, where it controls lamellipodia formation in uPAR-transfected HEK293 cells adhering to vitronectin. In this respect, the engineered constraint in uPARH47C-N259C thus bypasses the regulatory role of uPA binding, resulting in a constitutively active uPAR. In conclusion, our data argue for a biological relevance of the interdomain dynamics of the glycolipid-anchored uPAR on the cell surface.

Expression of C4.4A, a structural uPAR homolog, reflects squamous epithelial differentiation in the adult mouse and during embryogenesis.

The glycosylphosphatidylinositol (GPI)–anchored C4.4A was originally identified as a metastasis-associated protein by differential screening of rat pancreatic carcinoma cell lines. C4.4A is accordingly expressed in various human carcinoma lesions. Although C4.4A is a structural homolog of the urokinase receptor (uPAR), which is implicated in cancer invasion and metastasis, no function has so far been assigned to C4.4A. To assist future studies on its function in both physiological and pathophysiological conditions, the present study provide a global survey on C4.4A expression in the normal mouse by a comprehensive immunohistochemical mapping. This task was accomplished by staining paraffin-embedded tissues with a specific rabbit polyclonal anti-C4.4A antibody. In the adult mouse, C4.4A was predominantly expressed in the suprabasal layers of the squamous epithelia of the oral cavity, esophagus, non-glandular portion of the rodent stomach, anus, vagina, cornea, and skin. This epithelial confinement was particularly evident from the abrupt termination of C4.4A expression at the squamo-columnar transition zones found at the ano-rectal and utero-vaginal junctions, for example. During mouse embryogenesis, C4.4A expression first appears in the developing squamous epithelium at embryonic day 13.5. This anatomical location of C4.4A is thus concordant with a possible functional role in early differentiation of stratified squamous epithelia.

Ly6/uPAR-related protein C4.4A as a marker of solid growth pattern and poor prognosis in lung adenocarcinoma.

Introduction: We have recently shown that the protein C4.4A is induced in early precursor lesions of pulmonary adenocarcinomas and squamous cell carcinomas. In the present study, we aimed at analyzing the impact of C4.4A on the survival of non–small cell lung cancer patients and determining whether its unexpected expression in adenocarcinomas could be attributed to a specific growth type (lepidic, acinar, papillary, micropapillary, solid). Methods: Sections from the center and periphery of the primary tumor, as well as N2-positive lymph node metastases, were stained by immunohistochemistry for C4.4A and scored semi-quantitatively for intensity and frequency of positive tumor cells. Results: C4.4A score (intensity × frequency) in the tumor center was a highly significant prognostic factor in adenocarcinomas (n = 88), both in univariate (p = 0.004; hazard ratio [95% confidence interval] = 1.44 [1.12–1.85]) and multivariate statistical analysis (p = 0.0005; hazard ratio = 1.65 [1.24–2.19]), demonstrating decreasing survival with increasing score. In contrast, C4.4A did not provide prognostic information in squamous cell carcinomas (n = 104). Pathological stage was significant in both groups. In the adenocarcinomas, C4.4A expression was clearly associated with, but a stronger prognostic factor than, solid growth. Conclusions: The present results substantiate the potential value of C4.4A as a prognostic marker in pulmonary adenocarcinomas seen earlier in a smaller, independent patient cohort. Importantly, we also show that C4.4A is a surrogate marker for adenocarcinoma solid growth. Recent data suggest that C4.4A is negatively regulated by the tumor suppressor liver kinase B1, which is inactivated in some adenocarcinomas, providing a possible link to the impact of C4.4A on the survival of these patients.

Expression of C4.4A in precursor lesions of pulmonary adenocarcinoma and squamous cell carcinoma

The protein C4.4A, a structural homologue of the urokinase‐type plasminogen activator receptor, is a potential new biomarker in non‐small cell lung cancer, with high levels of expression recently shown to correlate to poor survival of adenocarcinoma patients. In this study, C4.4A immunoreactivity in precursor lesions of lung squamous cell carcinoma and adenocarcinoma was investigated by stainings with a specific anti‐C4.4A antibody. In the transformation from normal bronchial epithelium to squamous cell carcinoma, C4.4A was weakly expressed in basal cell hyperplasia but dramatically increased in squamous metaplasia. This was confined to the cell membrane and sustained in dysplasia, carcinoma in situ, and the invasive carcinoma. The induction of C4.4A already at the stage of hyperplasia could indicate that it is a marker of very early squamous differentiation, which aligns well with our earlier finding that C4.4A expression levels do not provide prognostic information on the survival of squamous cell carcinoma patients. In the progression from normal alveolar epithelium to peripheral adenocarcinoma, we observed an unexpected, distinct cytoplasmic staining for C4.4A in a fraction of atypical adenomatous hyperplasias, while most bronchioloalveolar carcinomas were negative. Likewise, only a fraction of the invasive adenocarcinomas was positive for C4.4A. With a view to the prognostic impact of C4.4A in adenocarcinoma patients, this finding might suggest that C4.4A could be an early biomarker for a possibly more malignant subtype of this disease.

C4.4A gene ablation is compatible with normal epidermal development and causes modest overt phenotypes

C4.4A is a modular glycolipid-anchored Ly6/uPAR/α-neurotoxin multidomain protein that exhibits a prominent membrane-associated expression in stratified squamous epithelia. C4.4A is also expressed in various solid cancer lesions, where high expression levels often are correlated to poor prognosis. Circumstantial evidence suggests a role for C4.4A in cell adhesion, migration, and invasion, but a well-defined biological function is currently unknown. In the present study, we have generated and characterized the first C4.4A-deficient mouse line to gain insight into the functional significance of C4.4A in normal physiology and cancer progression. The unchallenged C4.4A-deficient mice were viable, fertile, born in a normal Mendelian distribution and, surprisingly, displayed normal development of squamous epithelia. The C4.4A-deficient mice were, nonetheless, significantly lighter than littermate controls predominantly due to differences in fat mass. Congenital C4.4A deficiency delayed migration of keratinocytes enclosing incisional skin wounds in male mice. In chemically induced bladder carcinomas, C4.4A deficiency attenuated the incidence of invasive lesions despite having no effect on total tumour burden. This new C4.4A-deficient mouse line provides a useful platform for future studies on functional aspects of C4.4A in tumour cell invasion in vivo.

C4.4A as a biomarker in pulmonary adenocarcinoma and squamous cell carcinoma.

The high prevalence and mortality of lung cancer, together with a poor 5-year survival of only approximately 15%, emphasize the need for prognostic and predictive factors to improve patient treatment. C4.4A, a member of the Ly6/uPAR family of membrane proteins, qualifies as such a potential informative biomarker in non-small cell lung cancer. Under normal physiological conditions, it is primarily expressed in suprabasal layers of stratified squamous epithelia. Consequently, it is absent from healthy bronchial and alveolar tissue, but nevertheless appears at early stages in the progression to invasive carcinomas of the lung, i.e., in bronchial hyperplasia/metaplasia and atypical adenomatous hyperplasia. In the stages leading to pulmonary squamous cell carcinoma, expression is sustained in dysplasia, carcinoma in situ and invasive carcinomas, and this pertains to the normal presence of C4.4A in squamous epithelium. In pulmonary adenocarcinomas, a fraction of cases is positive for C4.4A, which is surprising, given the origin of these carcinomas from mucin-producing and not squamous epithelium. Interestingly, this correlates with a highly compromised patient survival and a predominant solid tumor growth pattern. Circumstantial evidence suggests an inverse relationship between C4.4A and the tumor suppressor LKB1. This might provide a link to the prognostic impact of C4.4A in patients with adenocarcinomas of the lung and could potentially be exploited for predicting the efficacy of treatment targeting components of the LKB1 pathway.

Structure and Inhibition of the Urokinase-Type Plasminogen Activator Receptor

The urokinase-type plasminogen activator receptor (uPAR/CD87) is a glycolipid-anchored receptor that is involved in focalizing plasminogen activation to the cell surface due to its high-affinity binding to the urokinase-type plasminogen activator (uPA). This chapter describes recent accomplishments in the molecular understanding of the structural biology of uPAR and its interactions with the cognate ligands, uPA and vitronectin. Furthermore, the structural basis for the pharmacological inhibition of uPAR by monoclonal antibodies, recombinant fusion proteins, and synthetic peptide antagonists are discussed. These compounds may prove valuable as drug candidates in combined intervention strategies targeting tumor invasion and metastasis.