CD13 regulation of membrane recycling: implications for cancer dissemination

ABSTRACT

Membrane recycling is critical to numerous cell functions and its dysregulation contributes to cancer and metastasis. We established that activation of the transmembrane molecule aminopeptidase N (ANPEP, also known as CD13) tethers the IQ motif containing, guanosine triphosphate hydrolase activating protein 1 (IQGAP1) scaffolding protein at the plasma membrane, thus stimulating the recycling regulator ADP-ribosylation factor 6 (ARF6) to ensure proper recycling of β1-integrin and other membrane components impacting cell attachment.

Cell attachment to the extracellular matrix, followed by activation of the integrin transmembrane receptor and downstream signal transduction are key steps in adhesion-dependent growth and cell migration.¹ These cellular processes are highly dependent upon well regulated, tightly controlled cascades of events involving internalization of membrane vesicles harboring adhesion and signaling molecules and their timely return to the cell surface to generate platforms for signal transduction and cell-extracellular matrix (ECM) attachment.^2,3 Vesicular trafficking is regulated by numerous scaffolding proteins such as guanosine triphosphate hydrolases (GTPases), regulatory GTPase exchange factors (GEFs) and GTPase activating proteins (GAPs), the exocyst tethering complex and transmembrane anchoring proteins. These orchestrate the journey through various endosomal compartments, from the early endosomes to perinuclear recycling endosomes ultimately recycling back to the plasma membrane.⁴ Logically, interruption of any of the steps in the trafficking process will impact pathways controlling cell attachment and growth factor signaling involved in homeostasis of the cell.

Aminopeptidase N (ANPEP, also known as CD13) is a transmembrane metalloprotease that is highly expressed in immune cells and activated endothelial cells at the sites of inflammation as well as certain epithelial cells and fibroblasts.⁵ Our novel observations have clearly shown that CD13 is a surprisingly multifunctional molecule that often works independently of its enzymatic activity to regulate diverse processes such as inflammatory cell adhesion, trafficking and signal transduction; antigen presentation, cell-matrix adhesion, cell-cell adhesion; endothelial invasion, migration and angiogenesis; skeletal muscle repair and maintenance of satellite stem cell populations; receptor-mediated endocytosis and endocytic trafficking; oxidative stress and apoptosis and focal adhesion turnover, most likely by virtue of its ability to regulate endocytosis and membrane organization. We have demonstrated that in endothelial cells CD13 is an important regulator of angiogenesis that controls uptake of the pro-angiogenic peptide bradykinin and its bradykinin receptor 2 (B₂R) to promote endothelial cell invasion and motility.⁶ In addition, we showed that in macrophages and dendritic cells, CD13 is activated by Src-kinase phosphorylation of tyrosine 6 in its cytoplasmic tail to become a novel regulator of dynamin-mediated endocytosis of various receptors such as toll-like receptor 4 (TLR4), mannose receptor and transferrin receptor. We further showed that CD13-dependent internalization of these receptors impacts downstream signal transduction pathways to regulate numerous disparate processes that rely on endocytosis, including cell adhesion and motility.⁷

In our recently published investigation,⁸ we explored a potential endocytic regulatory role for CD13 in the context of the well-characterized program of integrin endosomal trafficking and cell migration (Figure 1). We showed that in wild type (WT) murine embryonic fibroblasts (MEFs), CD13 and β1-integrin co-internalize, traffic to Early Endosome Antigen 1+ (EEA1+) and Ras-associated binding protein 5+ (Rab5+) early endosomes and recycle to the plasma membrane together via Ras-associated binding protein 11+ (Rab11a+) recycling endosomes. Conversely in CD13-deficient MEFs, internalized β1-integrin is again found initially in the early endosomes but rather than recycling, β1-integrin traffics from early endosomes to the Ras-associated binding protein 7+ (Rab7+) late endosomes/lysosomes and is eventually degraded. Pulse-chase assays in a human epithelial cell line engineered to express human CD13 or a phosphorylation-defective mutant, where CD13 tyrosine 6 is mutated to phenylalanine (CD13Y6F), confirmed CD13’s role in recycling and that this process required CD13 phosphorylation at this site. Functionally, the absence of CD13 led to reduced cell spreading, adhesion and cell-ECM migration in a wound-healing assay, consistent with reduced β1-integrin expression.

Figure 1.

Aminopeptidase N (ANPEP, also known as CD13) in receptor recycling. (a) In wild type (WT) cells, β1-integrin and CD13 phosphorylated on tyrosine 6 (CD13^Y6) co-internalize into early endosomes, continue to recycling endosomes and recycle to the plasma membrane, enabling cell-extracellular matrix (ECM) adhesion and migration. (b) Alternatively, cells lacking CD13 or expressing an inactive CD13 phosphorylation mutant again internalize β1-integrin into early endosomes but it is aberrantly sorted to lysosomes where it is ultimately degraded. Mechanistically, CD13 tethers a complex containing the scaffolding protein IQ motif containing, guanosine triphosphate hydrolase activating protein 1 (IQGAP1), α-actinin, active ADP-ribosylation factor 6 (ARF6), the guanosine exchange factor for ARF6 (EFA6), and β1-integrin at the plasma membrane to allow proper β1-integrin recycling and cell migration. In the absence of CD13 no active ARF6 is detected in the plasma membrane and IQGAP1 is not recruited to the migrating front, thereby diminishing cell adhesion, spreading and migration.

Extending our earlier observation that CD13-mediated cell-cell adhesion depends on its interaction with the scaffolding protein, IQ motif containing, guanosine triphosphate hydrolase activating protein 1 (IQGAP1),⁹ we analyzed human anti-CD13 monoclonal antibody immunoprecipitated complexes from cell lines expressing WT, CD13Y6F or vector control for the presence of the β1-integrin recycling regulator, ADP-ribosylation factor 6, (ARF6). We found IQGAP1 and active ARF6 in the CD13 complex only in cells expressing WT CD13 but not the empty vector or the CD13Y6F mutant, while expression of the hemagglutinin antigen (HA)-tagged, inactive ARF6 mutant where threonine 27 is mutated to asparagine (ARF6-T27N) did not associate with WT CD13, implying that CD13 only interacts with the active GTP-bound form of ARF6. Furthermore, specifically blocking ARF6 activity with the amino-terminal effector domain inhibitory peptide in WT cells recapitulated the loss of β1-integrin cell surface expression in cells lacking CD13 or expressing the inactive phosphorylation mutant CD13Y6F, further supporting a direct regulation of ARF6 by CD13. Therefore, in WT cells, a complex containing activated CD13, β1-integrin, IQGAP1, active-ARF6, its GEF, exchange factor for ARF6 (EFA6) and filamentous actin localizes at the leading edge of migrating cells and is required to maintain active ARF6 to regulate migration. Deficiency of CD13 reduced accumulation of these proteins at the migrating front, suggesting that CD13 is necessary to tether this complex at the cell membrane to maintain ARF6 activation, promote proper β1-integrin endosomal trafficking and thus is a critical regulator of cell-ECM interactions and membrane composition.

Interestingly, complexes containing analogous components have been shown to regulate trafficking and recycling of membrane microdomains to the plasma membrane to maintain anchorage-dependent growth. In healthy cells, integrin attachment to the ECM activates Ras-related protein A (RalA), stimulating ARF6 to deliver raft microdomain components that organize signaling molecules to enhance receptor–effector interactions, promoting signal transduction and cell growth.¹⁰ However, in tumor cells expressing oncogenic Harvey rat sarcoma viral oncogene homolog (H-ras), both RalA and ARF6 were constitutively activated regardless of cell attachment, allowing anchorage-independent growth, a hallmark of cancer. Similarly, changes in ECM adhesion affected by CD13-dependent trafficking of adhesion molecules would promote cell detachment and metastatic spread. Thus, increasing our understanding of the molecular mechanisms regulating fundamental cell biological processes such as vesicular trafficking and membrane organization will enable the rational design of future cancer therapeutics.

Funding Statement

This work was supported by the National Heart, Lung, and Blood Institute [R01HL125186]; National Heart, Lung, and Blood Institute [R01HL127449].