Identification of Cytoplasmic Motifs Required for Short Prolactin Receptor Internalization

doi:10.1074/jbc.272.11.7062

Identification of Cytoplasmic Motifs Required for Short Prolactin Receptor Internalization*

From ^‡ INSERM Unité 344, Endocrinologie Moléculaire, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris, Cedex 15, France and
** Systèmes Moléculaires et Biologie Structurale, Laboratoire de Mineralogie-Cristallographie, Universités Paris VI et Paris VII, CNRS URA 09, 4 Place Jussieu, 75252 Paris, Cedex 05, France

^‡‡ To whom correspondence should be addressed. Tel.: 33-1-40-61-53-10; Fax: 33-1-43-06-04-43; E-mail: kelly{at}necker.fr

↵^∥ Current address: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.

Abstract

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 β-turn structure (amino acids 276-279). We propose these two motifs are involved in PRLR endocytosis. Finally, we show that α-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.

Footnotes

↵^§ Recipient of a fellowship from Roussel-Uclaf Company and Association pour la Recherche sur le Cancer.
↵^¶ Supported by a fellowship from the European Economic Community.
↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

TR

transferrin receptor

PRL

prolactin

PRLR

PRL receptor

GH

growth hormone

aa

amino acid(s)

oPRL

ovine PRL

hGH

human GH

mAb

monoclonal antibody

CHO

Chinese hamster ovary

DMEM

Dulbecco's modified Eagle's medium

PAGE

polyacrylamide gel electrophoresis

BSA

bovine serum albumin

PBS

phosphate-buffered saline

HBB

HEPES binding buffer.
↵² M. Applanat, O. Gualillo, A. Pezet, V. Vincent, M. Edery, and P. A. Kelly, submitted for publication.
- Received October 1, 1996.