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The epidermis is highly sensitive to retinoids, and vitamin A (retinol) is a critical factor in the regulation of skin cell differentiation and proliferation. Despite extensive knowledge of retinoid-mediated gene transcription effects on epidermal cells and evidence for retinoid-mediated suppression of carcinogenesis in skin, basic transport events, especially cellular uptake, of this vitamin remain poorly understood and controversial. Herein, evidence is presented for receptormediated uptake of retinol-binding protein, RBP, the specific circulatory vitamin A carrier, in the A431 human epidermal cell line. Cellular RBP uptake was significantly inhibited by anti-RBP IgG. Addition of transthyretin (TTR), a circulatory protein that can interact with RBP, to the internalization assay also significantly reduced RBP uptake to 49.4±4.6% (± SEM) of control values (p<0.01). RBP uptake was impaired by sucrose, a known inhibitor of early endocytosis, but not significantly affected by a disruptor of later trafficking events, chlorpromazine. Binding analysis indicated saturable RBP binding to the cell surface and a total of about 94,000 binding sites/cell. Based on dissociation constants, two RBP binding sites were detected with a 50-fold affinity difference: 0.7 and 35.0 nM, with 12,000 and 82,000 receptors/cell, respectively. These results indicate that high affinity RBP receptors capable of internalizing RBP independently of TTR exist in these malignant keratinocytes, and that TTR influences binding of RBP to its putative receptor(s). Overall, the data establish membrane transport parameters for RBP, and provide a basis for examining modulation of vitamin A endocytosis that may accompany changes in proliferation or differentiation state of epidermal cells. Introduction Biologically active derivatives of vitamin A (retinol) such as 9-cis and all-trans-retinoic acid are involved in a wide range of physiological regulatory processes. At the molecular and cellular level, this regulation involves control of cell proliferation and differentiation through retinoid-dependent effects on gene expression (reviewed in refs. 1-4). These effects are critical for maintaining skin health and suppressing carcinogenesis in skin (1,2,4-6). Despite extensive knowledge of various retinoid-dependent gene expression effects in epidermal and other cell types, however, much less is known about retinoid transport events at the cell membrane; and endocytosis of circulatory vitamin A carrier proteins by epidermal cells or epidermoid cancer cells remains poorly understood and controversial (see below). A specific extracellular transport system for retinol exists in humans and other vertebrates and is mediated by the circulatory retinol-binding protein (RBP) (reviewed in refs. 3,7,8). This protein, of about 20 kDa, is a member of the lipocalin protein family. A fraction of RBP in the circulation is found associated with a homotetrameric protein, transthyretin (TTR) (reviewed in refs. 3,7,8). TTR is also one of the circulatory carries of thyroid hormones. The retinol-RBP-TTR and retinol-RBP complexes are the specific human circulatory retinol transport complexes (less specific transporters such as albumin and lipoproteins can also carry some forms of vitamin A) (reviewed in refs. 3,7,8). The possible roles of RBP and TTR in mediating cellular retinol uptake are not well defined. There is controversy regarding cell membrane receptors for RBP or TTR and receptor-independent cellular uptake mechanisms for this vitamin. In several tissues and cell types, cell membrane receptors for RBP or TTR have been reported based on binding studies, and both are candidates for mediating the endocytosis of retinol (reviewed in ref. 3; see Discussion for additional references). A controversy exists regarding cell surface RBP receptors in keratinocytes. There is evidence both for (9,10) and against (11,12) the existence of such receptors. In the present study, we provide evidence for an RBP receptor in A431 cells, a human cutaneous squamous cell carcinoma cell line. We characterize several basic transport parameters including affinity of the receptor-ligand interaction, number of cell surface binding sites, and sensitivity to inhibition by several endocytic transport modulators. Furthermore, our INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 17: 627-631, 2006 627 Evidence for a specific cell membrane retinol-binding protein transport mechanism in a human keratinocyte line JANET HUANG and AMANDIO VIEIRA Endocrine and Metabolic Research Laboratory, Faculty of Applied Sciences and Institute for Health Research (IHRE), Simon Fraser University, Burnaby, BC, Canada Received September 26, 2005; Accepted November 24, 2005 _________________________________________ Correspondence to: Dr Amandio Vieira, Endocrine and Metabolic Research Laboratory, Faculty of Applied Sciences, Simon Fraser University, K9600, 8888 University Drive, Burnaby, BC V5A 1S6,