Major histocompatibility complex (MHC) class I and MHC class I-type molecules such as the neonatal Fcgamma-receptor, FcRn, are heterodimers consisting of a transmembrane alpha-chain non-covalently associated with beta2-microglobulin (beta2m). Human placental villous syncytiotrophoblast (STB) lacks MHC class I molecules, but express hFcRn that mediates materno-fetal transmission of immunoglobulin G (IgG). Trophoblast-derived BeWo cells that are used to study placental IgG transport likewise express beta2m and low levels of hFcRn alpha-chain. The contribution of FcRn alpha-chain in retention and subcellular distribution of beta2m in STB and BeWo cells is unclear. To investigate this issue, we increased expression of hFcRn alpha-chain in BeWo cells (BeWo/hFcRn) by cDNA transfection. Overexpressed hFcRn protein exhibited the characteristic pH-dependent IgG binding and association with beta2m. In comparison to parental BeWo cells, beta2m mRNA levels in BeWo/hFcRn cells were not significantly altered, but total cell-associated beta2m protein was increased by 120%. Treatment of BeWo and BeWo/hFcRn cells with brefeldin A, an inhibitor of the secretory pathway, abrogated this effect, demonstrating that hFcRn alpha-chain expression retained otherwise secreted beta2m. Flow cytometry revealed that beta2m plasma membrane expression was unaffected by alpha-chain overexpression whereas by fluorescence microscopy a preferential staining of beta2m in peripheral endosomes was observed.

Calmodulin is a ubiquitous calcium-binding protein involved in the regulation of a variety of calcium-dependent cell functions. The present study provides evidence for a role of calmodulin in several steps of membrane transport along the endocytic pathway. Treatment of Madin-Darby canine kidney cells expressing the polymeric immunoglobulin receptor or a macrophage IgG Fc receptor with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) inhibited their ability to mediate transcytosis of dimeric IgA or lysosomal degradation of immune complexes, respectively. Since both pathways rely on intact microtubules, the inhibitory effect of W-7 may reflect an effect on calmodulin-regulated microtubule function. However, although W-7 did not affect endocytosis, the drug also inhibited recycling of receptors from apical or basolateral endosomes back to the respective surfaces. Thus, calmodulin may regulate microtubule-dependent and independent endocytic membrane transport pathways.