Prolactin stimulates milk protein promoter in CHO cells cotransfected with prolactin receptor cDNA

Lesueur, Laurence; Edery, Marc; Paly, J.; Clark, J. F.; Kelly, Paul A.; Djiane, Jean

doi:10.1016/0303-7207(90)90079-n

Cited by 60 publications

(31 citation statements)

References 10 publications

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“…It has been shown that PRLR stably expressed in CHO cells exhibit cellular localization, binding, and functional characteristics similar to those found in wild type PRL target cells (37,38).…”

Section: Methodsmentioning

confidence: 84%

Identification of Cytoplasmic Motifs Required for Short Prolactin Receptor Internalization

Vincent

Goffin

Rozakis-Adcock

et al. 1997

Journal of Biological Chemistry

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Cloning of rat prolactin receptor (PRLR) cDNAs revealed the existence of two isoforms, termed short and long according to the length of their cytoplasmic domain. Internalization studies show, first, that PRLR internalization is hormone-dependent and, second, that ligand-receptor complexes of the short PRLR are internalized to a larger extent compared to the long form. In order to identify regions within the cytoplasmic domain of the short PRLR required for efficient internalization, serial truncations of the cytoplasmic tail were performed by inserting a stop codon in place of those encoding residues 282, 273, 262, 253, 244, or 237 (wild type short PRLR contains 291 amino acids). Our data show that two motifs, lying within residues 253-261 and 273-281, are involved in internalization. Both regions contain a consensus feature identified within other receptors as internalization signals, namely a di-leucine peptide (amino acids 259 -260) and a tetrapeptide predicted to adopt a ␤-turn structure (amino acids 276 -279). We propose these two motifs are involved in PRLR endocytosis. Finally, we show that ␣-adaptin, a component of adaptor protein AP-2, coprecipitates with short PRLR complexes upon PRL stimulation, which strongly suggests that PRLR internalization is mediated by the clathrin-coated pits endocytotic pathway.

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Section: Methodsmentioning

confidence: 84%

Identification of Cytoplasmic Motifs Required for Short Prolactin Receptor Internalization

Vincent

Goffin

Rozakis-Adcock

et al. 1997

Journal of Biological Chemistry

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“…In this subclone, an examination of binding affinity showed that the PRL receptor exhibited a K a ]4 nM 1 . These CHO-transfected cells responded to PRL by stimulating the cotransfected milk protein gene promoter, proving that such cells were fully capable of transmitting the PRL signal and that PRL-R was functional (Lesueur et al 1990(Lesueur et al , 1991. The cells were grown in Ham's F12 medium (Seromed, Strasbourg, France) supplemented with 10% (v/v) fetal calf serum (Gibco, Grand Island, NY, USA), -glutamine (2·5 nM, Sigma, L'Isle d'Abeau, Chesnes, France) and 1% penicillin-streptomycin (Gibco).…”

Section: Cell Culturesmentioning

confidence: 92%

“…We used CHO cells transfected with the long form of the rabbit PRL receptor cDNA (CHO-E32), as previously described (Lesueur et al 1990, Bignon et al 1993. In this subclone, an examination of binding affinity showed that the PRL receptor exhibited a K a ]4 nM 1 .…”

Section: Cell Culturesmentioning

confidence: 99%

Prolactin induces an inward current through voltage-independent Ca2+ channels in Chinese hamster ovary cells stably expressing prolactin receptor

Ratovondrahona¹,

Fahmi²,

Fournier³

et al. 1998

Journal of Molecular Endocrinology

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There is still only limited understanding of the early steps of prolactin (PRL) signal transduction in target cells. Recent studies have identified some of the essential first steps: these include the rapid association of the PRL receptor with JAK tyrosine kinases and tyrosine phosphorylation of a number of proteins, including members of the signal transducer and activator of transcription (Stats) family. On the other hand, binding of PRL to its receptor is rapidly followed by calcium influx. However, PRL-induced ionic events and the related ionic channels involved have not been clearly established.This work was undertaken to characterise the channels responsible for calcium influx and to obtain an insight into their activation processes. Using the patch-clamp technique in the cellattached configuration, single Ca 2+ channel currents were recorded following PRL application (10 nM) in Chinese hamster ovary (CHO) cells stably expressing PRL receptor (CHO-E32). Statistical analysis showed that the recorded currents were voltage-independent, with a slope conductance of 16 pS. Although these channels were present in excised patches, the fact that PRL was unable to activate them suggested that a soluble cytoplasmic component may be required. Application of the purified inositol phosphate, Ins(1,3,4,5)P4 (2 µM), to the inside of the excised patch membrane activated the voltage-independent 16 pS Ca 2+ channel. The open probability (Popen) was enhanced. The inositol phosphates Ins(1,2,3,4,5)P5 and Ins(1,4,5)P3 did not affect channel activity while InsP6 (20 µM) had some effect, although less marked than that of Ins(1,3,4,5)P4. Using the anion-exchange HPLC technique, we then studied the effects of PRL (10 nM) on the turnover of inositol phosphates (InsPs) in CHO-E32. Our studies showed that PRL induces rapid increases in the production of Ins(1,3,4,5)P4 (207% at 30 s), InsP5 (171% at 30 s), and InsP6 (241% at 30 s). Conversely, Ins(1,4,5)P3 showed a transient decrease at 5 s, accompanied by a concomitant increase in Ins(1,3,4,5)P4, suggesting that the former could be transiently phosphorylated to produce the latter. Comparison of the production kinetics of Ins(1,4,5)P3, Ins(1,3,4,5)P4, InsP5, and InsP6 indicated the possibility of additional metabolic routes which have yet to be determined. This study suggests that PRL promotes Ca 2+ entry through voltage-independent Ca 2+ channels that may be activated by Ins(1,3,4,5)P4 and InsP6.

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“…The availability of full-length coding sequences for l-and s-oPRLR provided us with an opportunity to functionally test signal transduction partners (ligand, receptors, target promoter) of the same species (sheep), making this cellular system more physiologically relevant than that we used before (Lesueur et al 1990). In addition, the remaining components of the signaling pathway were endogenous, avoiding ligand-independent activations of Jak2 (Lebrun et al 1995a, Gao et al 1996 or of STAT5 (Moriggl et al 1996) due to overexpression.…”

Section: Discussionmentioning

confidence: 99%

“…The next day, the cells were starved for 16 h by incubation in GC3 medium. On the third day, the cells were transfected with lipofectamine (Gibco, France), following the manufacturer's instructions, by PCH110, a plasmid encoding the -gal activity (Pharmacia), and either by pBJ23, a plasmid bearing the ( 4000 to +40) ovine -lactoglobulin promoter-CAT reporter (for CAT assay) (Lesueur et al 1990), or by LHRE-tkluc, a luciferase reporter plasmid made of six repeats of rat -casein STAT5-responsive sequence, upstream of a thymidine kinase minimal promoter linked to a luciferase reporter gene and, when indicated, by l-oPRLRpLKneo, s-oPRLR-pLKneo or rbPRLR-pLKneo. Transfected cells were then incubated for 24 (luciferase assay) or 72 (CAT assay) h in the presence (two plates) or absence (two plates) of 400 ng/ml oPRL in GC3 medium containing 10 6 M dexamethasone.…”

Section: Chloramphenicol Acetyl Transferase (Cat)mentioning

confidence: 99%

In vitro expression of long and short ovine prolactin receptors: activation of Jak2/STAT5 pathway is not sufficient to account for prolactin signal transduction to the ovine beta-lactoglobulin gene promoter

Bignon¹,

Daniel²,

Belair³

et al. 1999

Journal of Molecular Endocrinology

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The recent finding that sheep had long (l-oPRLR) and short (s-oPRLR) prolactin receptors provided new tools to further explore prolactin signaling to target genes. Here we used CHO cells transfected with l-oPRLR or s-oPRLR cDNAs to compare the activation of known key steps of prolactin signaling by the two receptors. We found that prolactin stimulated l-oPRLR tyrosine phosphorylation, although it lacked the last tyrosine residue found in other long prolactin receptors. In addition, l-oPRLR and s-oPRLR both responded to prolactin stimulation by (1) Janus kinase 2 (Jak2) tyrosine phosphorylation, (2) DNA-binding activation of signal transducer and activator of transcription 5 (STAT5), (3) stimulation of transcription from a promoter made of six repeats of STAT5-responsive sequence. However, although it contains STAT5-binding consensus sequences, the ovine -lactoglobulin promoter ( 4000 to +40) was transactivated by l-oPRLR, but not by s-oPRLR. Taken together, our results indicate that activation of Jak2/STAT5 pathway alone is not sufficient to account for prolactin-induced transcription of this milk protein gene, and that sequences of its promoter, other than STAT5-specific sequences, account for the opposite transcriptional activation capabilities of l-oPRLR and s-oPRLR.

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Prolactin stimulates milk protein promoter in CHO cells cotransfected with prolactin receptor cDNA

Cited by 60 publications

References 10 publications

Identification of Cytoplasmic Motifs Required for Short Prolactin Receptor Internalization

Identification of Cytoplasmic Motifs Required for Short Prolactin Receptor Internalization

Prolactin induces an inward current through voltage-independent Ca2+ channels in Chinese hamster ovary cells stably expressing prolactin receptor

In vitro expression of long and short ovine prolactin receptors: activation of Jak2/STAT5 pathway is not sufficient to account for prolactin signal transduction to the ovine beta-lactoglobulin gene promoter

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