The Lutheran antigens are recently characterized glycoproteins in which the extracellular region contains five immunoglobulin like domains, suggesting some recognition function. A recent abstract suggests that the Lutheran glycoproteins (Lu gps) act as erythrocyte receptors for soluble laminin (Udani, M., Jefferson, S., Daymont, C., Zen, Q., and Telen, M. J. (1996) Blood 88, Suppl. 1, 6 (abstr.)). In the present report, we provided the definitive proof of the laminin receptor function of the Lu gps by demonstrating that stably transfected cells (murine L929 and human K562 cell lines) expressing the Lu gps bound laminin in solution and acquired adhesive properties to laminin-coated plastic dishes but not to fibronectin, vitronectin, transferrin, fibrinogen, or fibrin. Furthermore, expression of either the long-tail (85 kDa) or the short-tail (78 kDa) Lu gps, which differ by the presence or the absence of the last 40 amino acids of the cytoplasmic domain, respectively, conferred to transfected cells the same laminin binding capacity. We also confirmed by flow cytometry analysis that the level of laminin binding to red cells is correlated with the level of Lu antigen expression. Indeed, Lunull cells did not bind to laminin, whereas sickle cells from most patients homozygous for hemoglobin S overexpressed Lu antigens and exhibited an increased binding to laminin, as compared with normal red cells. Laminin binding to normal and sickle red cells as well as to Lu transfected cells was totally inhibited by a soluble Lu-Fc chimeric fragment containing the extracellular domain of the Lu gps. During in vitro erythropoiesis performed by two-phase liquid cultures of human peripheral blood, the appearance of Lu antigens in late erythroid differentiation was concomitant with the laminin binding capacity of the cultured erythroblasts. Altogether, our results demonstrated that long-tail and short-tail Lu gps are adhesion molecules that bind equally well laminin and strongly suggested that these glycoproteins are the unique receptors for laminin in normal and sickle mature red cells as well as in erythroid progenitors.

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We have performed a detailed analysis of the cis‐acting sequences involved in the erythroid‐specific expression of the human glycophorin B (GPB) promoter and found that this promoter could be divided into two regions. The proximal region, −1 to −60, contains a GATA binding sequence around −37 and an SP1 binding sequence around −50. This region is active in erythroid and non‐erythroid cells. The distal region, −60 to −95, contains two overlapping protein binding sites around −75, one for hGATA‐1 and one for ubiquitous proteins. This distal region completely represses the activity of the proximal promoter in non‐erythroid cells and defines the −95 GPB construct as a GPB promoter that displays erythroid specificity. Using site directed mutagenesis, we show that the −37 GATA and the −50 SP1 binding sites are necessary for efficient activity of the −95 GPB construct. Mutations that impair the −75 GATA‐1 binding result in extinction of the −95 GPB construct activity if the −75 ubiquitous binding site is not altered, or in loss of erythroid specificity if the −75 ubiquitous binding site is also mutated. Using a cotransfection assay, we found that hGATA‐1 can efficiently activate transcription of the −95 GPB construct in non‐erythroid cells. This transactivation is abolished by mutations that impair either the −37 GATA‐1 or the −50 SP1 binding. Mutations that impair the −75 GATA‐1 binding and still allow the −75 ubiquitous binding also abolish the transactivation of the −95 GPB construct, indicating that hGATA‐1 can remove repression of the GPB promoter by displacement of the ubiquitous proteins.(ABSTRACT TRUNCATED AT 250 WORDS)