While the C-terminal cytoplasmic tail of anion exchanger 1 (AE1, band 3) has been reported to possess important physiological roles, including one for proper membrane trafficking, its precise characteristics remain unclear. To clarify the overall structural consequences of the conserved sequence EL(K/Q)(L/C)LD(A/G)DD, containing the core binding sequence LDADD for carbonic anhydrase II, in the C-terminal region, we analyzed the membrane expression and turnover of bovine AE1 with a series of truncation and substitution mutations in HEK293 cells. Immunofluorescence microscopy and cell-surface biotinylation demonstrated that truncation mutants missing 18 C-terminal residues targeted the plasma membrane, but the one lacking the conserved region, by truncation of 28 amino acid residues, was retained inside the cells. Substitutions of Ala for Glusup(901), Leusup(902), Leusup(905), and Aspsup(906) in the sequence E901L(K/Q)(L/C)LDADD909 of bovine AE1 or those in the corresponding murine sequence also caused intracellular retention, though these mutants had half-lives comparable to that for wild-type AE1. These data demonstrate that the conserved amino acid residues Glusup(1), Leusup(2), Leusup(5), and Aspsup(6) in the EL(K/Q)(L/C)LD(A/G)DD region have essential structural consequences in stable expression of AE1 at the plasma membrane regardless of the ability in binding to carbonic anhydrase II of this region.

We examined the effect of stroma-derived factors, hepatocyte growth factor (HGF) and leptin, on morphological differentiation of bovine mammary epithelial cells (BMEC) in collagen gel three-dimensional culture in vitro. BMEC treated with HGF, but not leptin, formed duct-like organoids. The formation of organoids by HGF was enhanced by treatment with a mixture of insulin, cortisol and prolactin, while BMEC treated with the mixture alone did not produce the organoid. In contrast, the formation of organoids by HGF was dose-dependently inhibited by simultaneous addition of leptin, regardless of the presence or absence of the hormone mixture. These results suggest that stroma-derived factors intricately regulate mammary epithelial morphogenesis.