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The absence of a direct route to the apical plasma membrane (PM) for single transmembrane domain (TMD) proteins in polarized hepatic cells has been inferred but never directly demonstrated. The genes encoding three pairs of apical PM proteins, whose extracellular domains are targeted exclusively to the apical milieu in Madin-Darby canine kidney cells, were packaged into recombinant adenovirus and delivered to WIF-B cells in vitro and liver hepatocytes in vivo. By immunofluorescence and pulse-chase metabolic labeling, we found that the soluble constructs were overwhelmingly secreted into the basolateral milieu, which in vivo is the blood and in vitro is the culture medium. The full-length proteins were first delivered to the basolateral surface but then concentrated in the apical PM. Our results imply that hepatic cells lack trans-Golgi network (TGN)-based machinery for directly sorting single transmembrane domain apical proteins and raise interesting questions about current models of PM protein sorting in polarized and nonpolarized cells.

Downstream signaling triggered by the binding of fibroblast growth factor-2 (FGF2) to its tyrosine-kinase receptors involves the activation of mitogen-activated protein kinase kinase (MEK) with consequent phosphorylation of extracellular signal-regulated kinases (ERKs). Here we demonstrate that FGF2 induces ERK1/2 activation in bovine aortic endothelial (BAE) cells and that the continuous presence of the growth factor is required for sustained ERK1/2 phosphorylation. This is prevented by the MEK inhibitors PD 098059 and U0126, which also inhibit FGF2-mediated upregulation of urokinase-type plasminogen activator (uPA) and in vitro formation of capillary-like structures in three-dimensional type I collagen gel. Various FGF2 mutants originated by deletion or substitution of basic amino acid residues in the amino terminus or in the carboxyl terminus of FGF2 retained the capacity to induce a long-lasting activation of ERK1/2 in BAE cells. Among them, K128Q/R129Q-FGF2 was also able to stimulate uPA production and morphogenesis whereas R129Q/K134Q-FGF2 caused uPA upregulation only. In contrast, K27, 30Q/R31Q-FGF2, K128Q/K138Q-FGF2 and R118,129Q/K119,128Q-FGF2 exerted a significant uPA-inducing and morphogenic activity in an ERK1/2-dependent manner only in the presence of heparin. Furthermore, no uPA upregulation and morphogenesis was observed in BAE cells treated with the deletion mutant (delta)27-32-FGF2 even in the presence of soluble heparin. Thus, mutational analysis of FGF2 dissociates the capacity of the growth factor to induce a persistent activation of ERK1/2 from its ability to stimulate uPA upregulation and/or in vitro angiogenesis. In conclusion, the data indicate that ERK1/2 phosphorylation is a key step in the signal transduction pathway switched on by FGF2 in endothelial cells. Nevertheless, a sustained ERK1/2 activation is not sufficient to trigger uPA upregulation and morphogenesis. FGF2 mutants may represent useful tools to dissect the signal transduction pathway(s) mediating the complex response elicited by an angiogenic stimulus in endothelial cells.