The addition of a glycosyl-phosphatidylinositol-anchor to a soluble form of neutral endopeptidase re-establishes its apical targeting in LLC-PK1 cells.

Neutral endopeptidase (NEP, EC 3.4.24.11) is a type I1 membrane protein that possesses a large extracellular domain anchored to the plasma membrane by a N-terminal transmembrane spanning domain (for review see [ 11). Various molecular forms of NEP have been produced that differ in the type of anchoring mechanism(s) used to attach the protein to the membrane (reviewed in [2]). Briefly, a soluble form of NEP (sNEP) was produced by replacing the transmembrane region and short cytoplasmic domain with a cleavable signal peptide [3]. A glycosyl-phosphatidylinositol (GP1)-anchored form of NEP (GPINEP) was created by the addition of a GPI-anchor signal attachment sequence to the soluble form [4]. NEP is found on a variety of mammalian cell types. The protein is abundantly expressed at the apical surface of epithelial cens, in particular those of the choroid plexus, lining of the small intestine and renal cortex. In an attempt to understand the targeting and sorting mechanisms in polarised epithelia we have studied different molecular forms of NEP expressed in various epithelial cell lines (for review see [5]). When NEP was expressed in the pig kidney proximal tubule cell line, LLC-PKI, it was shown to be directly targeted to the apical surface [6]. However, when sNEP was expressed in LLC-PKI cells an unpolarised secreted distribution of the protein was observed. Here we report on the effect the addition of a GPI-anchor has on the targeting of the NEP ectodomain (sNEP) in LLC-PKI cells. The GPI-NEP encoding region of the vector pSVGPINEP [4] was subcloned into the pRcCMV vector (Invitrogen, CA) downstream of the cytomegalovb promotor and enhancer elements to give pRcCMVGPINEP. This vector was then transfected into LLC-PK1 cells using the CaP04 precipitation method and selection undertaken using the neomycin analog, (3418. in accordance with the protocol previously described for NEP [6] . Evidence for the presence of the GPI-anchor in GPI-NEP was obtained by labelling cells with ['HI ethanolamine; a component of the core structure of the GPI-anchor. GPI-NEP was shown, by fluorography, to incorporate ['HI ethanolamine. Control samples, from LLC-PK1 cells expressing NEP, did not incorporate [3H] ethanolamine. Conformation that GPI-NEP was indeed anchored via a GPI-anchor came from studies using phosphatidylinositolspecific phospholipase C (PI-PLC). PI-PLC specifically cleaves GPI-anchored proteins and can liberate them from cellular membranes [7]. Membranes, from LLC-PKI cells expressing GPINEP. were subjected to PI-PLC and NEP immunoreactive material was released. This was not the case when using membranes from LLC-PKI cells expressing wild-type NEP. In order to assess the polarity of GPI-NEP, cells were grown on filters and proteins from both the apical and basolateral surfaces