Abstract: The cation-independent mannose 6-phosphate receptor (CI-MPR), also known as the IGF2 receptor or CD222, is a multifunctional type I transmembrane glycoprotein ubiquitously expressed in most eukaryotic cell types. Through the receptor’s ability to bind a variety of unrelated extracellular and intracellular ligands, it is involved in a wide array of functions including protein trafficking, lysosomal biogenesis, internalization, regulation of cell growth, cell migration and apoptosis. CI-MPR has a large extracellular region comprised of 15 contiguous domains, four of which interact with phosphorylated glycans on lysosomal enzymes. Here we present a series of biophysical studies, along with crystal structures, providing information on how the N-terminal 5 domains of this receptor work in concert to bind and release carbohydrates. High-resolution electron microscopy as well as hydroxyl radical protein footprinting (HRPF) of this multifunctional multidomain construct demonstrates dynamic conformational changes occur as a consequence of ligand binding and different pH conditions, These data, coupled with surface plasmon resonance studies and molecular modeling, allow us to propose a bi-dentate oligosaccharide binding model, which could explain how high affinity carbohydrate binding is achieved through allosteric domain cooperativity.

The cation-independent mannose 6-phosphate receptor (CI-MPR, IGF2 receptor or CD222), is a multifunctional glycoprotein required for normal development. Through the receptor's ability to bind unrelated extracellular and intracellular ligands, it participates in numerous functions including protein trafficking, lysosomal biogenesis, and regulation of cell growth. Clinically, endogenous CI-MPR delivers infused recombinant enzymes to lysosomes in the treatment of lysosomal storage diseases. Although four of the 15 domains comprising CI-MPR's extracellular region bind phosphorylated glycans on lysosomal enzymes, knowledge of how CI-MPR interacts with ~60 different lysosomal enzymes is limited. Here, we show by electron microscopy and hydroxyl radical protein footprinting that the N-terminal region of CI-MPR undergoes dynamic conformational changes as a consequence of ligand binding and different pH conditions. These data, coupled with X-ray crystallography, surface plasmon resonance and molecular modeling, allow us to propose a model explaining how high-affinity carbohydrate binding is achieved through allosteric domain cooperativity.