The Rabbit Mammary Gland Prolactin Receptor Is Tyrosine-phosphorylated in Response to Prolactin in Vivo and in Vitro

doi:10.1074/jbc.270.10.5136

The Rabbit Mammary Gland Prolactin Receptor Is Tyrosine-phosphorylated in Response to Prolactin in Vivo and in Vitro(*)

From the Unite d'Endocrinologie Moleculaire, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex, France and Physiology and Pharmacology Department and the Centre for Molecular and Cellular Biology, University of Queensland, St. Lucia, Queensland 4072, Australia

^§ To whom correspondence should be addressed. Tel.: 61-7-3652607; Fax: 61-7-3651766.

Abstract

We report the first in vivo study demonstrating tyrosine phosphorylation of mammary gland proteins including the prolactin receptor, in response to the injection of prolactin. Immunoblotting of mammary gland membrane extracts revealed that subunits of 200, 130, 115, 100, 90, 70, and 45 kDa display increased tyrosine phosphorylation within 5 min of prolactin administration. The 100-kDa component was identified as the full-length prolactin receptor by a variety of means including immunoprecipitation and immunoblotting with monoclonal (U5, 917, 110, and 82) and polyclonal (46) antibodies to the prolactin receptor. Maximal receptor phosphorylation was seen within 1 min of hormone injection, and to obtain a strong response it was necessary to deprive rabbits of their endogenous prolactin for 36 h. Rapid tyrosine phosphorylation of the full-length receptor was verified by its demonstration in Chinese hamster ovary cells stably transfected with rabbit prolactin receptor cDNA. Both in vivo and in vitro, the phosphorylation signal was transient, being markedly reduced within 10 min of exposure to prolactin. Tyrosine-phosphorylated receptor was shown to be associated with JAK 2 by immunoblotting of receptor immunoprecipitated from transfected Chinese hamster ovary cells with polyclonal 46. A 48-kDa ATP-binding protein was also shown to be associated with the mammary gland receptor by U5 or polyclonal 46 immunoprecipitation of receptor complexes following covalent labeling with [α-P]azido-ATP.

Our demonstration of prolactin receptor tyrosine phosphorylation raises the possibility of signaling pathways regulated by receptor/SH2 protein interaction, which would facilitate prolactin specific responses. The fact that a period of hormone deprivation is needed for significant hormone triggered receptor phosphorylation indicates that the mammary gland receptor exists in a largely desensitized state in vivo, analogous to the related growth hormone receptor.

Footnotes

↵* This work was supported by a grant from the French Ministere de la Recherche et de la Technologie (to M. J. W. during sabbatical leave in France) and by a grant from INSERM (Contrat de Recherche Externe 930703). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

GH

growth hormone

CHO

Chinese hamster ovary

JAK

Janus kinase

mAb

monoclonal antibody

MAP

microtubule-associated protein

oPrl

ovine prolactin.
↵²N. Daniel, M. J. Waters, C. Bignon, and J. Djiane, unpublished data.
↵³V. Mitev and M. J. Waters, unpublished results.
- Received July 29, 1994.
- Revision received November 18, 1994.