Mechanisms of apical protein sorting in polarized thyroid epithelial cells Concetta Lipardi, Lucio Nitsch, Chiara Zurzolo* Centro di Endocrinologia ed Oncologia Sperimentale del CNR-Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università degli Studi di Napoli ‘Federico II’, Via Pansini 5, 80131 Naples, Italy (Received 6 July 1998; accepted 30 November 1998) Abstract — The process leading to thyroid hormone synthesis is vectorial and depends upon the polarized organization of the thyrocytes into the follicular unit. Thyrocyte membrane proteins are delivered to two distinct domains of the plasma membrane using apical (AP) and basolateral (BL) sorting signals. A recent hypothesis for AP sorting proposes that apically destined proteins cluster with glycosphingolipids (GSLs) and cholesterol, into microdomains (or rafts) of the Golgi membrane from which AP vesicles originate. In MDCK cells the human neurotrophin receptor, p75 hNTR , is delivered to the AP surface through a sorting signal, rich in O-glycosylated sugars, identified in its ectodomain. We have investigated whether this signal is functional in the thyroid-derived FRT cell line and whether p75 hNTR clusters into lipid rafts to be sorted to the AP membrane. We found that p75 hNTR is apically delivered via a direct pathway and does not associate with rafts during its transport to the surface of FRT cells. Therefore, although the same signal could be recognized by different cell types thyroid cells may possess a tissue-specific sorting machinery. © Société française de biochimie et biologie moléculaire / Elsevier, Paris thyroid polarity / lipid raft / p75 hNTR / apical sorting / FRT cell line 1. Introduction The functional and morphological unit of the thyroid gland is the follicle. This is formed by a single layer of thyrocytes [1] which, like all epithelial cells, have their plasma membrane divided by tight junctions in two domains, the apical (AP) and basolateral (BL) domains, with distinct protein and lipid composition [1–3]. While the AP domain faces the follicular lumen into which the newly synthesized thyroglobulin is secreted and accumu- lated, thereby serving as the substrate for iodination and hormonogenesis [4, 5], the basolateral domain faces the interstitium/bloodstream from which thyrocytes take up iodide and release thyroid hormones [6–8]. The process leading to thyroid hormone secretion is vectorial and depends upon the polarized organization of the thyrocytes. The establishment and maintenance of this organization, and subsequently its functionality, is ensured by the continuous and regulated intracellular sorting of different lipids and proteins to the cell surface [9]. In the model system of MDCK cells, derived from dog kidney, it has been shown that plasma membrane proteins are exported to the Golgi complex where, at the trans-Golgi network (TGN), they are separated into AP and BL vesicles [10] through specific sorting information. While BL sorting signals have been identified as short amino acid sequences within the cytoplasmic tails of several transmembrane proteins [11, 12], sorting to the AP surface involves more than one determinant. The current model for AP sorting proposes that, within the Golgi bilayer, glycosphingolipids (GSLs) and cholesterol cluster into microdomains, which function as sorting platforms (so- called ‘rafts’) for proteins destined to the AP surface [13]. The rafts are insoluble in Triton X-100 (TX-100) at 4 °C and are able to float into lighter fractions on sucrose density gradients. Glycosylphosphatidyl inositol (GPI)- anchored proteins possibly use their GPI-anchor to cluster with rafts and to be apically delivered [14, 15]. Associa- tion with rafts and AP sorting can also be mediated by the transmembrane domains, as shown in the cases of the influenza virus proteins neuraminidase [16] and hemag- glutinin [17]. In addition, the mannose-rich core of N-glycans or a juxtamembrane region rich in O-glycosylated residues are involved in AP sorting of secretory and/or transmembrane proteins [18, 19]. AP and BL sorting signals are decoded by a specific molecular machinery. Defects in their recognition create abnormalities in protein transport and may specifically contribute, in the case of thyroid gland, to different pathologies [20]. For example, a defect in the targeting of thyroglobulin to the AP extracellular space may lead to metabolic disturbances, abnormal development, and goi- ter [21]. Furthermore, loss of epithelial cell polarity, and therefore alteration of protein sorting to the surface, is an * Correspondence and reprints Abbreviations: AP, apical; BL, basolateral; TX-100, Triton X-100; GSLs, glycosphingolipids; GPI-anchored proteins, glycosylphosphatidylinositol-anchored proteins; TSH, thyrotro- pin; p75 hNTR , p75 human neurotrophin receptor. Biochimie 81 (1999) 347-353 © Société française de biochimie et biologie moléculaire / Elsevier, Paris