The mannose 6-phosphate receptor cytoplasmic domain is not sufficient to alter the cellular distribution of a chimeric EGF receptor.
Unlike most receptors, 300 kd mannose 6-phosphate receptors (MPRs) are localized primarily in the trans-Golgi network (TGN) and endosomes, and they cycle constitutively between these compartments. Yet, when present at the cell surface, MPRs are internalized together with other cell surface receptors in clathrin-coated vesicles. We constructed a chimeric receptor, comprised of human EGF receptor extracellular and transmembrane domains joined to the bovine MPR cytoplasmic domain, to test whether the MPR cytoplasmic domain contained sufficient information to direct a cell surface receptor into both of these transport pathways. The expressed protein was stable, bound EGF with high affinity, and was efficiently endocytosed and recycled back to the cell surface, in the presence or absence of EGF. If the cytoplasmic domain alone is responsible for sorting native MPRs, chimeric receptors might have been expected to be located primarily in the TGN and in endosomes at steady state. Surprisingly, under conditions in which essentially all endogenous MPRs were intracellular, greater than 85% of the chimeric receptors were located at the cell surface. These experiments demonstrate that the MPR cytoplasmic domain is not sufficient to alter the distribution of the EGF receptor, and suggest a role for extracellular and transmembrane domains in MPR routing.
Dintzis
S M
SM
Department of Biochemistry, Stanford University School of Medicine, CA 94305-5307.
Pfeffer
S R
SR
D000818:Animals; D001692:Biological Transport; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D007163:Immunosorbent Techniques; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection
D007140:Immunoglobulin Fab Fragments; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors