INTRODUCTION Cell sites of signal transduction, after the binding of a hormone to its receptor, have been believed to occur mainly at the plasma membrane. However, the internalisation of ligand- receptor complexes into the endosomal apparatus may be consistent with intracellular signalling (Baas et al., 1995; Bevan et al., 1996). As prolactin (PRL) is internalised in lactating mammary epithelial cells (MEC) and transcytosed to the lumen (Ollivier-Bousquet, 1998), these cells provide a model to study whether, during its transport through the cell, PRL initiates distinct signalling cascades that lead to different biological responses. PRL exerts many biological actions through the prolactin receptors (PRLR), such as the control of casein gene expression (Hennighausen et al., 1997) and the in vitro stimulation of protein secretion, which is referred to as the secretagogue effect (Ollivier-Bousquet, 1978). Transduction of the PRL message within the cell from the cell membrane to the nucleus, which causes activation of the transcription of milk protein genes, includes oligomerisation of the receptor and activation of the self-phosphorylating Janus kinase 2 (Jak2), which in turn phosphorylates tyrosine residues on the receptor and the signal transducers and activators of transcription (STATs), Stat5a and Stat5b. These factors dimerise, translocate to the nucleus and then bind to specific promoter elements on PRL-responsive genes (Hennighausen et al., 1997; Goffin and Kelly, 1997). However, molecular events that lead to the transduction of the secretagogue effect of PRL remain partly unknown. They include a very rapid and transient release of arachidonic acid within a few minutes of the addition of PRL, and the metabolism of this polyunsaturated fatty acid (PUFA) (Blachier et al., 1988). Moreover, arachidonic acid, when added to the incubation medium of lactating MEC, stimulates casein secretion, probably after being metabolised in products of the lipoxygenase pathway (Ollivier-Bousquet, 1982; Ollivier-Bousquet, 1984). Together these results strongly suggest that prolactin stimulates casein secretion by the release of arachidonic acid. In contrast to hormones that are endocytosed and then rapidly degraded in lysosomes (Burgess et al., 1992), PRL is carried through the MEC by transcytosis and then released in the milk in either intact or cleaved molecular forms (Sinha, 1995). PRL is transported via a vesicular pathway that includes not only endosomes, late endosomes and multivesicular bodies, but also vesicles located in the Golgi region and secretory vesicles containing casein micelles (Ollivier-Bousquet, 1998). The transcytosis of PRL can be slowed down or inhibited in tissues incubated at low temperatures (25°C) for 1 hour (Seddiki et al., 1991), or in mammary tissues from rats that receive a PUFA-deficient diet (Ollivier-Bousquet et al., 1993; Ollivier-Bousquet et al., 1997). Under these two conditions, in which PRL accumulates in late endosomes and multivesicular bodies, basal secretion is not affected but the secretagogue effect of the hormone is inhibited (Seddiki et al., 1991; Ollivier-Bousquet et al., 1993; Ollivier-Bousquet et al., 1997). One question that arises is whether the journey of PRL or the 1883 Prolactin added to the incubation medium of lactating mammary epithelial cells is transported from the basal to the apical region of cells through the Golgi region and concomitantly stimulates arachidonic acid release and protein milk secretion. We report that when PRL is added after disorganisation of the Golgi apparatus by brefeldin A treatment, prolactin signalling to expression of genes for milk proteins and prolactin endocytosis are not affected. However, prolactin transport to the apical region of cells (transcytosis), as well as prolactin-induced arachidonic acid release and subsequent stimulation of the secretion of caseins, which are located in a post-Golgi compartment, are inhibited. This inhibition was not a consequence of damage to the secretory machinery, as under the same conditions, protein secretion could be stimulated by the addition of arachidonic acid to the incubation medium. Thus, it is possible to discriminate between prolactin-induced actions that are dependent (signalling to milk protein secretion) or independent (signalling to milk gene expression) on the integrity of the Golgi apparatus. These results suggest that these two biological actions may be transduced via distinct intracellular pathways, and support the hypothesis that prolactin signals may be emitted at various cellular sites. Key words: Prolactin, Lactating mammary epithelial cells, Brefeldin A, Golgi apparatus, Secretion, Prolactin receptor, Signal transduction SUMMARY Prolactin signalling to milk protein secretion but not to gene expression depends on the integrity of the Golgi region Mustapha Lkhider 1 , Barbara Pétridou 2 , Alain Aubourg 2 and Michèle Ollivier-Bousquet 2, * 1 Faculté des Sciences, UCD, El Jadida, Maroc 2 Unité de Biologie Cellulaire et Moléculaire, INRA, 78352 Jouy-en-Josas Cedex, France *Author for correspondence (e-mail: ollivier@biotec.jouy.inra.fr) Accepted 1 March 2001 Journal of Cell Science 114, 1883-1891 © The Company of Biologists Ltd RESEARCH ARTICLE