Differential involvement of the Ras and Rap1 small GTPases in vasoactive intestinal and pituitary adenylyl cyclase activating polypeptides control of the prolactin gene.
In pituitary cells, transcriptional regulation of the prolactin (PRL) gene and prolactin secretion are controlled by multiple transduction pathways through the activation of G protein coupled receptors and receptor tyrosine kinases. In the somatolactotrope GH4C1 cell line, we have previously identified crosstalk between the MAPKinase cascade ERK1/2 and the cAMP/protein kinase A pathway after the activation of the VPAC2 receptor by vasoactive intestinal polypeptide (VIP) or pituitary adenylyl cyclase-activating polypeptide (PACAP38). In the present study, we focus on the involvement of the GTPases Ras and Rap1 as downstream components of signal transmission initiated by activation of the VPAC2 receptor. By using pull-down experiments, we show that VIP and PACAP38 preferentially activate Rap1, whereas thyrotropin releasing hormone (TRH) and epidermal growth factor (EGF) mainly activate Ras GTPase. Experiments involving the expression of the dominant-negative mutants of Ras and Rap1 signaling (RasN17 or Rap1N17) indicate that both GTPases Ras and Rap1 are recruited for the ERK activation by VIP and PACAP38, whereas Rap1 is poorly involved in TRH or EGF-induced ERK activation. The use of U0126, a selective inhibitor of MAPKinase kinase, provides evidence that MAPKinase contributes to the regulation of the PRL gene. Moreover, cotransfection of RasN17 or Rap1N17 with the PRL proximal promoter luciferase reporter construct indicates that Rap1 may be responsible for VIP/PACAP-induced activation of the PRL promoter. Interestingly, Ras would be involved as a negative regulator of VIP/PACAP-induced PRL gene activation, in contrast to its stimulatory role in the regulation of the PRL promoter by TRH and EGF.
Romano
David
D
Unité Mixte de Recherche 6544, Institut Fédératif de Recherche Jean-Roche, Faculté de Médecine Nord, 13916 Marseille Cedex 20, France.
Magalon
Karine
K
Ciampini
Audrey
A
Talet
Christine
C
Enjalbert
Alain
A
Gerard
Corinne
C
D000818:Animals; D045744:Cell Line, Tumor; D004815:Epidermal Growth Factor / Q000378:metabolism; D005786:Gene Expression Regulation; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009479:Neuropeptides / Q000378:metabolism*; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D010902:Pituitary Gland / Q000166:cytology; D011388:Prolactin / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D018005:Receptors, Vasoactive Intestinal Peptide / Q000378:metabolism; D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D015398:Signal Transduction; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism*; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D018631:ras Proteins / Q000378:metabolism* / Q000502:physiology
C495403:Adcyap1 protein, rat; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D018005:Receptors, Vasoactive Intestinal Peptide; D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D014660:Vasoactive Intestinal Peptide; D013973:Thyrotropin-Releasing Hormone; D004815:Epidermal Growth Factor; D011388:Prolactin; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020827:rap1 GTP-Binding Proteins; D018631:ras Proteins