Related Experiment Videos
Calmodulin binds to the basolateral targeting signal of the polymeric immunoglobulin receptor
S J Chapin1, C Enrich, B Aroeti
1Department of Anatomy, University of California, San Francisco 94143, USA.
Insights
Researchers identified the polymeric immunoglobulin receptor (pIgR) as a key calmodulin (CaM)-binding protein in rat liver endosomes. CaM binding to pIgR is Ca(2+)-dependent and occurs in the pIgR cytoplasmic tail, potentially regulating pIgR transcytosis and signaling.
Area of Science:
- Cell Biology
- Molecular Biology
- Protein Interactions
Background:
- Calmodulin (CaM) is a crucial calcium-binding protein involved in various cellular processes.
- The polymeric immunoglobulin receptor (pIgR) plays a vital role in transporting immunoglobulins across epithelial cells.
Purpose of the Study:
- To identify CaM-binding proteins in rat liver endosomes.
- To investigate the interaction between pIgR and CaM and its functional implications.
Main Methods:
- 125I-CaM overlays on two-dimensional protein blots.
- Immunostaining of blots.
- CaM-agarose binding assays with Madin-Darby canine kidney cells expressing pIgR.
- Chemical cross-linking experiments.
- Analysis of pIgR mutants.
Main Results:
- pIgR was identified as a major CaM-binding protein in rat liver endosomes.
- Detergent-solubilized pIgR binds CaM in a Ca(2+)-dependent and specific manner.
- The CaM binding site is in the membrane-proximal 17-amino acid segment of the pIgR cytoplasmic tail.
- CaM binding is not essential for pIgR basolateral targeting.
Conclusions:
- CaM specifically binds to pIgR in a Ca(2+)-dependent manner.
- CaM may regulate pIgR transcytosis and/or signaling.
- The pIgR cytoplasmic tail segment binds CaM and contains a basolateral targeting signal, but CaM binding is not required for this targeting.
Abstract:
We have identified a major calmodulin (CaM)-binding protein in rat liver endosomes using 125I-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. Furthermore, pIgR is the most prominent 35S-labeled CaM-binding protein in the detergent phase of Triton X-114-solubilized, metabolically labeled pIgR-expressing Madin-Darby canine kidney cells. CaM can be chemically cross-linked to both solubilized and membrane-associated pIgR, suggesting that binding can occur while the pIgR is in intact membranes. The CaM binding site is located in the membrane-proximal 17-amino acid segment of the pIgR cytoplasmic tail. This region of pIgR constitutes an autonomous basolateral targeting signal. However, binding of CaM to various pIgR mutants suggests that CaM binding is not necessary for basolateral targeting. We suggest that CaM may be involved in regulation of pIgR transcytosis and/or signaling by pIgR.