Mechanisms for High Affinity Mannose 6-Phosphate Ligand Binding to the Insulin-like Growth Factor II/Mannose 6-Phosphate Receptor: NEGATIVE COOPERATIVITY AND RECEPTOR OLIGOMERIZATION

James C Byrd,Richard G Macdonald

doi:10.1074/jbc.m000010200

James C Byrd, Richard G Macdonald

Open Access

https://doi.org/10.1074/jbc.m000010200

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Abstract

The two mannose 6-phosphate (Man-6-P) binding domains of the insulin-like growth factor II/mannose 6-phosphate receptor (Man-6-P/IGF2R), located in extracytoplasmic repeats 1-3 and 7-9, are capable of binding Man-6-P with low affinity and glycoproteins that contain more than one Man-6-P residue with high affinity. High affinity multivalent ligand binding sites could be formed through two possible mechanisms: the interaction of two Man-6-P binding domains within one Man-6-P/IGF2R molecule or by receptor oligomerization. To discriminate between these mechanisms, truncated FLAG epitope-tagged Man-6-P/IGF2R constructs, containing one or both of the Man-6-P binding domains, were expressed in 293T cells, and characterized for binding of pentamannose phosphate-bovine serum albumin (PMP-BSA), a pseudoglycoprotein bearing multiple Man-6-P residues. A construct containing all 15 repeats of the Man-6-P/IGF2R extracytoplasmic domain bound PMP-BSA with the same affinity as the full-length receptor (K(d) = 0.54 nm) with a curvilinear Scatchard plot. The presence of excess unlabeled PMP-BSA increased the dissociation rate of pre-formed (125)I-PMP-BSA/receptor complexes, suggesting negative cooperativity in multivalent ligand binding and affirming the role of multiple Man-6-P/IGF2R binding domains in forming high affinity binding sites. Truncated receptors containing only one Man-6-P binding domain and mutant receptor constructs, containing an Arg(1325) --> Ala mutation that eliminates binding to the repeats 7-9 binding domain, formed high affinity PMP-BSA binding, but with reduced stoichiometries. Collectively, these observations suggest that alignment of Man-6-P binding domains of separate Man-6-P/IGF2R molecules is responsible for the formation of high affinity Man-6-P binding sites and provide functional evidence for Man-6-P/IGF2R oligomerization.

Highlights

The two mannose 6-phosphate (Man-6-P) binding domains of the insulin-like growth factor II/mannose 6-phosphate receptor (Man-6-P/IGF2R), located in extracytoplasmic repeats 1–3 and 7–9, are capable of binding Man-6-P with low affinity and glycoproteins that contain more than one Man-6-P residue with high affinity
The sorting of newly synthesized lysosomal enzymes from the trans-Golgi network to pre-lysosomal acidic vesicles occurs through the recognition of mannose 6-phosphate (Man-6-P)1 markers by two membrane-bound mannose 6-phosphate receptors (MPRs), the cation-dependent Man-6-P receptor (CDMPR), and the insulin-like growth factor II/Man-6-P receptor (Man-6-P/IGF2R)
pentamannose phosphate-bovine serum albumin (PMP-BSA), a pseudoglycoprotein containing multiple Man6-P residues, was synthesized and iodinated for use as a multivalent ligand to measure the ability of the 15F construct to bind phosphomannosylated ligands

Summary

The abbreviations used are

Man-6-P, mannose 6-phosphate; IGF, insulin-like growth factor; Man-6-P/IGF2R, mannose 6-phosphate/ insulin-like growth factor II receptor; PMP, pentamannose phosphate; BSA, bovine serum albumin; CD-MPR, cation-dependent mannose 6-phosphate receptor; MPR, mannose 6-phosphate receptor; nt, nucleotide(s); PAGE, polyacrylamide gel electrophoresis; HBST, HEPESbuffered saline containing Triton X-100. Receptor oligomerization could result in the formation of two unique high affinity sites, which may explain observations of functional differences between the repeat 3 and repeat 9 Man-6-P binding domains with respect to their preference for sorting specific hydrolases [30]. Truncation of the Man-6-P/IGF2R, revealed that multiple regions outside of the minimal Man-6-P binding domains in repeats 3 and 9 play a role in both the formation of bivalent Man-6-P binding sites and the resultant affinity of these sites toward PMP-BSA These observations suggest that receptor oligomerization is the mechanism for the formation of high affinity Man-6-P binding in the extracytoplasmic domain of this receptor

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION