Calmodulin Binds to the Basolateral Targeting Signal of the Polymeric Immunoglobulin Receptor

Abstract

We have identified a major calmodulin (CaM)-binding protein in rat liver endosomes using 125 I-CaM overlays from two-dimensional protein blots. Immunostaining of blots demonstrates that this protein is the polymeric immunoglobulin receptor (pIgR). We further investigated the interaction between pIgR and CaM using Madin-Darby canine kidney cells stably expressing cloned wild-type and mutant pIgR. We found that detergent-solubilized pIgR binds to CaM-agarose in a Ca 2؉ -dependent fashion, and binding is inhibited by the addition of excess free CaM or the CaM antagonist W-13 (N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide), suggesting that pIgR binding to CaM is specific. The plasma membrane of polarized epithelial cells is divided into apical and basolateral surfaces, each having a distinct protein and lipid composition. Cells use two pathways to deliver proteins to the correct surface. First, newly made proteins are packaged in the trans-Golgi network (TGN) 1 into vesicles that deliver them directly to either the apical or basolateral surface. Second, proteins can be delivered first to one surface and then endocytosed and transcytosed to the opposite surface. At least in the well polarized Madin-Darby canine kidney (MDCK) cell line, targeting to the basolateral surface generally requires a sorting signal located in the cytoplasmic domain of the protein. For a number of basolateral proteins it has been found that mutations in the cytoplasmic domain prevent TGN to basolateral targeting (1). The first basolateral signal to be identified is the 17-amino acid membrane proximal segment (residues 653-670) of the cytoplasmic domain of the polymeric immunoglobulin receptor (pIgR) (2). This protein is normally delivered from the TGN to the basolateral surface, and then endocytosed and transcytosed to the apical surface. Deletion of most of this 17-residue segment prevents TGN to basolateral delivery. Moreover, this segment can be transplanted to a heterologous reporter molecule, which is then retargeted from the apical to the basolateral surface (2). In at least one other case, the low density lipoprotein receptor, autonomous basolateral sorting signals have been identified (3).The pIgR basolateral targeting signal has been systematically analyzed by alanine scanning mutagenesis (4). Mutation of His-656, Arg-657, or Val-660 to Ala substantially diminishes, but does not eliminate, TGN to basolateral targeting. Mutation of other residues had little or no effect. The structure of a synthetic 17-residue peptide corresponding to this signal has been determined by two-dimensional nuclear magnetic resonance spectroscopy (4). This peptide tends to adopt a putative type 1 ␤-turn, encompassing residues 658 -661, followed by a nascent helical structure. In MDCK cells polarized sorting takes place in both the TGN and after endocytosis. Mutations that diminish TGN to basolateral sorting and increase TGN to apical sorting have a similar effect on sorting in the endocytotic pathway, decreasing recycling to the basolateral surface and...

Membrane-permeable Calmodulin Inhibitors (e.g. W-7/W-13) Bind to Membranes, Changing the Electrostatic Surface Potential

“…Our results, however, suggest that this effect could equally well be due to W-7 reducing the surface potential of the plasma membrane, particularly because acidic lipids appear to mediate the membrane association of sphingosine kinase (70). An example of an integral membrane protein is the polymeric immunoglobulin receptor, pIgR, which has a 17-residue membrane-proximal cytoplasmic segment, residues 653-670, that binds Ca 2ϩ /CaM with high affinity (71). In the absence of Ca 2ϩ /CaM, this segment probably also binds to the negatively charged inner leaflet of the plasma membrane, since it has a net charge of ϩ5.…”

Dissection of the multifunctional “receptor-recycling” endocytic compartment of hepatocytes

“…Calmodulin emerges as a pivotal molecule for most of the functions accomplished in the heterogeneous ''recycling-endosomes''; it is involved in the recycling of transferrin and pIgR, in the transcytosis of pIgA, 60 in the endocytosis and recycling of ricin, 65 and it is also crucial in the regulation of several activities shown in caveolae. 66 It is remarkable that receptors for transferrin, 67 pIgA, 60,68 insulin, 69 and epidermal growth factor 70 are calmodulin-binding proteins. In 2 different procedures for the isolation of hepatic endosomes, calmodulin was shown to be highly and specifically concentrated in the receptor-enriched fraction.…”

Transferrin Receptor Functions as a Signal‐Transduction Molecule for its Own Recycling Via Increases in the Internal Ca2 Concentration

“…Similarly, if no Caz+ is present in the incubation medium, Tf-R recycling is inhibited, and this receptor is lost or inactivated in reticulocytes (Morgan, 1989). Conversely, an artificial elevation of Ca" stimulates apical transcytosis of transferrin (Chapin et al, 1996). We demonstrated previously that reticulocyte endocytic vesicles contain Ca'+-ATPase activity working under the regulation of a trimeric guanine-nucleotide-binding regulatory protein .…”