Distinct cytoplasmic regions of the prolactin receptor are required for prolactin-induced calcium entry.
Two cytoplasmic regions of the prolactin (PRL) receptor are well documented for their participation in PRL signal transduction, the membrane proximal box 1 and the COOH-terminal region. In order to study the role of these regions in PRL-induced Ca2+ increase, we use Chinese hamster ovary (CHO) cells stably transfected with mutated PRL receptor cDNA. These cells express the long form of PRL receptor deleted from box 1 (CHO Delta1 cells) or the 141 amino acids of the COOH-terminal region (CHO H3 cells). The patch-clamp technique in "whole-cell" configuration and microfluorimetric techniques were used singly or in combination. Data obtained for these cells were compared with those we have recently published using CHO cells expressing the wild-type long form of the PRL receptor (CHO TSE32). In contrast to CHO TSE32 cells, exposure of CHO Delta1 or H3 cells to PRL (0.05-50 nM) did not modify [Ca2+]i. We have previously shown that the PRL-induced calcium influx via voltage-insensitive, Ca2+ channels was due to the activation of tyrosine kinase-dependent K+ channels that hyperpolarize the CHO TSE32 cell membrane (hyperpolarization-driven Ca2+ influx). Therefore, two events are involved in PRL-induced Ca2+ changes (i) JAK2-activation of K+ channels and (ii) intracellular messenger-opening of Ca2+ channels. In CHO Delta1 cells, PRL (0.05-50 nM) neither hyperpolarized the membrane potential nor stimulated the JAK2-dependent K+ current, confirming the pivotal role played by box 1/JAK2 in the PRL-induced activation of K+ channels. However, when these cells were voltage-clamped below the resting membrane potential, application of 5 nM PRL resulted in an increase in Ca2+ influx. Therefore, box 1/JAK2 was not involved in the opening of these Ca2+ channels. In CHO H3 cells, 5 nM PRL activated the K+ current and hyperpolarized the membrane potential without any effect on [Ca2+]i. Moreover, PRL was also ineffective on CHO H3 cells voltage-clamped below the resting membrane potential. Therefore, the COOH-terminal region is involved in the production of the intracellular messenger that opens voltage-independent Ca2+ channels. We conclude from these findings that box 1 and COOH-terminal regions are both needed for PRL-induced Ca2+ changes.
Laboratoire de Neurophysiologie, Centre National de la Recherche Scientifique UMR 5543, Université de Bordeaux 2, 33076 Bordeaux Cédex, France.
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Sorin
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Goupille
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Vacher
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Paly
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Djiane
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Vacher
D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D016764:Cell Polarity; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D004553:Electric Conductivity; D005470:Fluorometry; D008564:Membrane Potentials / Q000187:drug effects; D016296:Mutagenesis; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000378:metabolism; D011388:Prolactin / Q000494:pharmacology*; D011817:Rabbits; D011981:Receptors, Prolactin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction; D014162:Transfection
D015220:Calcium Channels; D015221:Potassium Channels; D011981:Receptors, Prolactin; D011994:Recombinant Proteins; D011388:Prolactin; D002118:Calcium