M6PR Human

What is the basic structure of human M6PR?

Human cation-dependent mannose-6-phosphate receptor (CD-M6PR) is a 46 kDa type I transmembrane protein encoded by the M6PR gene. It consists of a single transmembrane domain with the C-terminus located on the cytoplasmic side of the lipid membrane . The mature protein is 277 amino acids in length, containing a 159-amino acid luminal region (residues 27-185) and a 67-amino acid cytoplasmic domain . The protein functions as a homodimer, with specific amino acid residues (Gln66, Arg111, Glu133, and Tyr143) that recognize carbohydrate moieties, while Asp103 interacts with divalent cations such as Mn²⁺ to increase ligand affinity and receptor oligomerization . The extracellular domain contains the mannose-6-phosphate (M6P) binding site that is crucial for its function in lysosomal enzyme transport.

How does human M6PR transport lysosomal enzymes?

Human M6PR functions as a sorter and deliverer of lysosomal enzymes through two primary pathways. In the first pathway, newly synthesized lysosomal enzymes bearing M6P markers bind to M6PR in the Golgi complex and are transported into the Golgi apparatus. In the second pathway, M6P-containing lysosomes in the extracellular matrix bind to M6PR on the cell membrane, facilitating transport of lysosomal enzymes . In both pathways, a receptor-ligand complex forms and is transported to lysosomes. In the acidic environment of lysosomes (low pH), the receptor-ligand complex dissociates, and M6PR is recycled back to the Golgi complex or plasma membrane . This system is critical for maintaining cellular metabolic and catabolic homeostasis through proper targeting of acid hydrolases to lysosomes .

What are the key differences between cation-dependent and cation-independent M6P receptors?

The human M6P receptor system comprises two distinct receptors: the cation-dependent M6P receptor (CD-M6PR, also known as the small M6PR) and the cation-independent M6P receptor (CI-M6PR). The CD-M6PR is a 46 kDa protein that requires divalent cations for optimal binding activity, showing enhanced ligand binding in the presence of cations like Mn²⁺ . In contrast, CI-M6PR is significantly larger and can bind ligands effectively regardless of cation presence. CI-M6PR also functions as the insulin-like growth factor II receptor, exhibiting multiple binding sites and broader ligand specificity . Research indicates that while both receptors participate in lysosomal enzyme trafficking, they serve complementary roles, with differential expression patterns and binding properties across tissues and developmental stages. CD-M6PR binding to mannose-6-phosphate shows a pH optimum between 6.0 and 6.5, which is important for its function in different cellular compartments .

What are the established techniques for purifying human M6PR for research?

The purification of human M6PR typically involves recombinant expression systems followed by multi-step chromatographic approaches. Researchers have developed stable cell lines expressing the receptor's extracellular domain, utilizing small molecule biomimetics of mannose-6-phosphate to aid in affinity purification . A methodological approach includes:

• Gene expression in mammalian cell lines (often HEK293 or CHO cells) for proper post-translational modifications

• Affinity chromatography using immobilized mannose-6-phosphate or synthetic analogs

• Size-exclusion chromatography to separate monomeric from dimeric forms

• Ion-exchange chromatography for final polishing

For detecting purified M6PR, Western blot analysis using specific antibodies such as anti-human CD-M6PR lumenal domain antibodies can verify the approximately 45 kDa protein band under reducing conditions . Researchers studying M6PR interactions can employ surface plasmon resonance to analyze binding kinetics with various ligands, including mannose-6-phosphate and synthetic analogs .

How can researchers effectively characterize M6PR binding kinetics and specificity?

Characterization of M6PR binding kinetics and specificity involves multiple complementary techniques:

• Surface Plasmon Resonance (SPR): This real-time, label-free technique allows determination of association (ka) and dissociation (kd) rate constants, as well as equilibrium dissociation constants (KD) for various ligands .

• Enzyme-Linked Immunosorbent Assay (ELISA): Useful for high-throughput screening of potential ligands and determining relative binding affinities.

• Cell-Based Binding Assays: Using cells overexpressing human M6PR, researchers can study binding under various pH and ionic conditions. Studies have shown that specific binding to human M6PR in permeabilized cells demonstrates a pH optimum between 6.0 and 6.5, with enhanced binding in the presence of divalent cations .

• Endocytosis Assays: Using enzymes like β-glucuronidase as traceable ligands, researchers can assess receptor-mediated endocytosis rates under different conditions. Data indicates that at pH 6.5, human M6PR mediates endocytosis effectively, while at pH 7.5, the rate drops to approximately 14% of that observed at the optimal pH .

When designing binding experiments, researchers should consider the influence of pH and cation concentration, as these significantly affect binding affinity. Controls should include competitive inhibition with free mannose-6-phosphate or analogs to confirm binding specificity.

What cell models are most appropriate for studying human M6PR function?

Several cell models have proven valuable for studying human M6PR function, each with specific advantages:

For investigating specific aspects of M6PR function, researchers have developed several experimental systems. For example, transfected mouse L cells that overexpress human M6PR but lack the insulin-like growth factor II/mannose 6-phosphate receptor have been used to study enzyme binding to cell surface receptors, binding to intracellular receptors in permeabilized cells, and receptor-mediated endocytosis of recombinant human β-glucuronidase . These systems allow for the isolation of CD-M6PR-specific effects from those mediated by CI-M6PR.

How does M6PR contribute to lysosomal enzyme sorting and trafficking?

M6PR plays a central role in the biosynthetic transport of newly synthesized acid hydrolases to lysosomes through the recognition of mannose-6-phosphate (M6P) residues. The process involves several coordinated steps:

• In the Golgi apparatus, newly synthesized lysosomal enzymes undergo post-translational modification to add M6P markers.

• M6PR binds these M6P-tagged enzymes in the trans-Golgi network (TGN).

• Receptor-enzyme complexes are packaged into clathrin-coated vesicles and transported to late endosomes.

• The acidic pH of endosomes causes dissociation of the enzymes from M6PR.

• M6PR is recycled back to the TGN via retrograde transport, while the enzymes continue to lysosomes .

Research using cells overexpressing human M6PR has demonstrated that these cells show reduced secretion of newly synthesized β-glucuronidase compared to control cells, indicating that overexpressed human M6PR can participate in sorting newly synthesized lysosomal enzymes and partially correct sorting defects in cells lacking the insulin-like growth factor II/mannose 6-phosphate receptor . This highlights M6PR's critical role in preventing inappropriate secretion of lysosomal enzymes.

What regulatory mechanisms control M6PR distribution and trafficking between cellular compartments?

M6PR distribution and trafficking between cellular compartments are regulated by multiple mechanisms:

• pH-Dependent Binding: M6PR-ligand interactions are highly pH-sensitive, with optimal binding occurring between pH 6.0-6.5 in the TGN, and dissociation occurring at the lower pH (≤5.5) of late endosomes, enabling cyclic receptor utilization .

• Cytoplasmic Tail Signals: The 67-amino acid cytoplasmic domain of CD-M6PR contains sorting signals that interact with adaptor proteins for clathrin-mediated vesicular transport. These signals direct the receptor's movement between the TGN, endosomes, and plasma membrane .

• Phosphorylation: Reversible phosphorylation of cytoplasmic residues modulates interaction with trafficking machinery components.

• Palmitoylation: This lipid modification affects the receptor's membrane association and trafficking properties.

• GTPase Regulation: Rab proteins, particularly Rab9, control M6PR retrieval from endosomes to the TGN.

Studies have shown that up to 2.3% of total functional receptor can be detected on the cell surface through enzyme binding assays . This surface pool participates in endocytosis of extracellular lysosomal enzymes, forming an alternative pathway for enzyme delivery to lysosomes. The distribution between intracellular compartments and the cell surface is dynamically regulated and can be altered in response to cellular conditions or in disease states.

How does M6PR interact with the broader endosomal-lysosomal system?

M6PR functions as a critical component within the broader endosomal-lysosomal system through multiple interactions:

• Adaptor Protein Complexes: M6PR interacts with AP-1, AP-2, and GGAs (Golgi-localized, γ-ear-containing, ARF-binding proteins) through its cytoplasmic tail to facilitate vesicular transport between different compartments.

• Retromer Complex: This protein complex recognizes and retrieves M6PR from endosomes, facilitating its return to the TGN.

• TIP47 (tail-interacting protein of 47 kDa): Binds specifically to the cytoplasmic domains of both M6P receptors, protecting them from degradation in late endosomes.

• ESCRT Machinery: While primarily involved in multivesicular body formation, the ESCRT system interacts with the endosomal-lysosomal pathway in which M6PR operates.

The endosomal-lysosomal system is fundamentally connected to numerous cellular processes, including autophagy, exosome formation, and plasma membrane repair . Dysfunction in this system has been implicated in various neurodegenerative disorders. M6PR's role in ensuring proper delivery of acid hydrolases to lysosomes is essential for maintaining the degradative and recycling functions of this system. Additionally, recent research has identified connections between mTORC1 signaling and M6PR transport, with mTORC1 regulating mannose-6-phosphate receptor trafficking through control of kinesin KIF13A, linking nutrient sensing to lysosomal function .

How is M6PR implicated in lysosomal storage diseases, and what therapeutic strategies target this pathway?

M6PR is fundamentally involved in lysosomal storage diseases (LSDs) due to its critical role in trafficking lysosomal enzymes. LSDs occur when lysosomal enzymes are absent, deficient, or dysfunctional, leading to accumulation of undigested macromolecules within lysosomes . Several therapeutic approaches targeting the M6PR pathway have been developed:

These therapeutic strategies leverage M6PR's natural role in lysosomal enzyme trafficking, aiming to bypass or overcome the pathological mechanisms of various LSDs.

What is known about M6PR's role in cancer biology and potential therapeutic targeting?

M6PR has been implicated in several aspects of cancer biology, with evidence suggesting both tumor-suppressive and oncogenic functions depending on the cancer type and context:

• Tumor Suppression: CI-M6PR (IGF2R) can sequester insulin-like growth factor II (IGF-II), preventing it from binding to the IGF-I receptor and activating mitogenic signaling pathways. Loss of M6PR expression has been observed in various cancers, supporting its potential tumor-suppressor role .

• Invasion and Metastasis: Research has shown that M6PR/IGF2R modulates the invasiveness of liver cells via its capacity to bind mannose 6-phosphate residues. Expression of insulin‐like growth factor-2 receptor has prognostic significance in human hepatocellular carcinoma and is influenced by transarterial chemoembolization .

• Lysosomal Enzyme Regulation: By controlling the trafficking of lysosomal proteases such as cathepsins, M6PR can influence tumor cell invasion and metastasis.

• Therapeutic Targeting: Several approaches leverage M6PR for cancer therapy:

  • Trichosanthin has been found to increase Granzyme B penetration into tumor cells by upregulating CI-MPR on the cell surface, enhancing cancer cell apoptosis .

  • Photodynamic therapy of prostate cancer cells has been improved through targeting of the cation-independent mannose 6-phosphate receptor .

  • M6PR can be utilized for selective delivery of cytotoxic agents to cancer cells that overexpress the receptor.

These findings highlight M6PR as both a prognostic marker and potential therapeutic target in various cancer types, with strategies focusing on either restoring its tumor-suppressive functions or exploiting its expression for targeted drug delivery.

How does M6PR function in immune system regulation and inflammation?

Emerging research has identified novel functions of M6PR in immune system regulation and inflammation:

• T-Cell Vulnerability: M6PR (CD222) has been identified as an emerging regulator of the immune system. Research has shown that mTORC1 regulates mannose-6-phosphate receptor transport and T-cell vulnerability to regulatory T cells by controlling kinesin KIF13A . This mechanism influences T-cell function and susceptibility to immunosuppression.

• TGF-β Activation: M6PR binds to latent TGF-β1 and facilitates its activation, thereby regulating this pleiotropic cytokine that plays crucial roles in immune tolerance and inflammation. Studies have demonstrated that cation-independent mannose 6-phosphate receptor inhibitors (e.g., PXS25) inhibit fibrosis in human proximal tubular cells by inhibiting conversion of latent to active TGF-β1 .

• Fibrosis Regulation: The mannose-6-phosphate analogue, PXS64, has been shown to inhibit fibrosis via the TGF-β1 pathway in human lung fibroblasts, indicating a role for M6PR in fibrotic disease processes .

• Hepatic Stellate Cell Targeting: Peptide-based siRNA nanocomplexes targeting hepatic stellate cells have been developed, leveraging M6PR for cell-specific delivery. This approach has been used for reduction of fibrogenesis by selective delivery of Rho kinase inhibitors to hepatic stellate cells in mice with liver injury .

These findings highlight M6PR's multifaceted role in immune regulation and its potential as a therapeutic target in inflammatory and fibrotic conditions. The receptor's expression on various immune cell populations and its involvement in cytokine processing make it a critical component of the immune regulatory network.

What structural determinants govern the specificity of human M6PR for different phosphomannosylated ligands?

• Binding Pocket Flexibility: How does the conformational flexibility of the binding pocket accommodate diverse phosphomannosylated structures? X-ray crystallography and cryo-EM studies of receptor-ligand complexes are needed to elucidate the structural dynamics during binding.

• Cooperative Binding: Does binding at one site influence binding at other sites within receptor dimers? Biophysical analyses using techniques like hydrogen-deuterium exchange mass spectrometry could reveal allosteric mechanisms.

• Synthetic Ligand Development: Research has explored several chemical approaches to develop synthetic M6PR ligands:

  • Sulfonate and phosphonate derivatives for cation-independent mannose 6-phosphate receptor targeting

  • Carboxylate analogues of the mannose 6-phosphate recognition marker

  • Phosphatase-inert "molecular rulers" to probe for bivalent mannose 6-phosphate ligand–receptor interactions

Future research should focus on developing high-resolution structures of human M6PR in complex with various ligands, combined with computational approaches to model binding dynamics. This could facilitate the rational design of more selective and potent synthetic ligands for therapeutic applications.

How do post-translational modifications of M6PR influence its trafficking and function in different cell types?

The functional diversity of M6PR across different tissues and cell types suggests cell-specific regulatory mechanisms, with post-translational modifications (PTMs) playing a key role:

• Phosphorylation Patterns: Different kinases phosphorylate specific serine and threonine residues in the cytoplasmic tail of M6PR, altering its interaction with adaptor proteins and trafficking machinery. Quantitative phosphoproteomics across cell types could reveal tissue-specific phosphorylation signatures.

• Palmitoylation: This reversible lipid modification affects membrane association and subdomain localization. How palmitoylation is regulated in response to cellular stress or differentiation state remains poorly understood.

• Glycosylation: M6PR itself undergoes N-glycosylation, but how this modification affects its folding, stability, and function across different cell types requires further investigation.

• Ubiquitination: This modification can influence receptor internalization and degradation rates. Cell-type-specific differences in the ubiquitination machinery may contribute to variable M6PR half-lives.

Methodologically, CRISPR-Cas9 gene editing to introduce mutation at specific PTM sites, combined with live-cell imaging of fluorescently tagged M6PR variants, could provide insights into how these modifications affect receptor dynamics in different cellular contexts. Additionally, mass spectrometry-based approaches can identify the complete PTM profile of M6PR isolated from different tissues and disease states.

What are the emerging connections between M6PR dysfunction and metabolic disorders?

Recent research has begun to uncover connections between M6PR function and metabolic regulation, particularly in the context of insulin resistance and metabolic syndrome:

• miRNA Regulation: Studies have shown that circulating miR-143-3p inhibition protects against insulin resistance in Metabolic Syndrome via targeting of the insulin-like growth factor 2 receptor. This suggests a regulatory role for M6PR in glucose metabolism .

• Circular RNA Interactions: CircRNF111 has been found to protect against insulin resistance and lipid deposition via regulating the miR-143-3p/IGF2R axis in Metabolic Syndrome, further supporting M6PR's involvement in metabolic regulation .

• Lysosomal Function in Metabolism: As the primary trafficking receptor for lysosomal enzymes, M6PR dysfunction could impair lysosomal degradation of lipids and glycogen, contributing to their accumulation in metabolic disorders.

• Growth Factor Signaling: Through its role in IGF-II binding (in the case of CI-M6PR), alterations in M6PR function may affect growth factor signaling pathways that regulate cellular metabolism.

Research methodologies to explore these connections should include:

• Tissue-specific knockout models to evaluate metabolic consequences of M6PR deficiency

• Metabolomic profiling of cells with altered M6PR expression or trafficking

• Integration of transcriptomic and proteomic data to identify molecular networks connecting M6PR to metabolic pathways

• Clinical studies correlating M6PR genetic variants or expression levels with metabolic parameters

These emerging connections suggest M6PR may be a novel therapeutic target for metabolic disorders, potentially through modulation of lysosomal function or growth factor signaling.