Differential distribution of mannose-6-phosphate receptors and furin in immature secretory granules.
In neuroendocrine cells sorting of proteins from immature secretory granules (ISGs) occurs during maturation and is achieved by clathrin-coated vesicles containing the adaptor protein (AP)-1. We have investigated the role of the mannose-6-phosphate receptors (M6PRs) in the recruitment of AP-1 to ISGs. M6PRs were detected in ISGs isolated from PC12 cells by subcellular fractionation, and by immuno-EM labelling on cryosections. In light of our previous results, where greater than 80% of the ISGs were found to contain furin, we examined the relationship between furin and M6PR on ISGs. By immunoisolation techniques we find that 50% at most of the ISGs contain the cation-independent (CI)-M6PR. Using sequential immunoisolation we could demonstrate that there are two populations of ISGs: those that have both M6PR and furin, and those which contain only furin. Furthermore, using immobilized GST-fusion proteins containing the cytoplasmic domain of the CI-M6PR we have shown binding of AP-1 requires casein kinase II phosphorylation of the CI-M6PR fusion protein, and in particular phosphorylation of Ser(2474). Addition of these phosphorylated GST-CI-M6PR fusion proteins to a cell-free assay reconstituting AP-1 binding to ISGs inhibits AP-1 recruitment to ISGs.
Dittié
A S
AS
Secretory Pathways Laboratory, Imperial Cancer Research Fund, London, WC2A 3PX, UK.
Klumperman
J
J
Tooze
S A
SA
D016475:3T3 Cells; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001667:Binding, Competitive; D047390:Casein Kinase II; D002417:Cattle; D003594:Cytoplasmic Granules / Q000378:metabolism* / Q000648:ultrastructure; D011992:Endosomes / Q000378:metabolism; D045683:Furin; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D006735:Horseradish Peroxidase / Q000493:pharmacokinetics; D006801:Humans; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D016716:PC12 Cells; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D017527:Receptor, IGF Type 2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D008565:Membrane Proteins; D017527:Receptor, IGF Type 2; D011993:Recombinant Fusion Proteins; D006735:Horseradish Peroxidase; D005982:Glutathione Transferase; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D013381:Subtilisins; D045683:Furin