Expression of the full-length rabbit prolactin receptor and its specific domains in baculovirus infected insect cells.
The prolactin receptor is a membrane protein mainly involved in the development of the mammary gland and in lactation in mammals. We used specific cDNA constructs and the insect/baculovirus expression system and produced independently and in large amounts several recombinant forms of the rabbit mammary gland prolactin receptor: the full-length receptor (L1, L2), a truncated membrane form (S), a secretable form of the extracellular domain (E) and two forms of the intracellular domain (I1, I2). Of these forms, the L1 and L2 are associated with the membrane fraction, the E is predominantly secreted into the medium and the I1 and I2 are expressed as soluble proteins and surprisingly, a great portion accumulates in the culture medium. The molecular mass (94 kDa) of the expressed full-length receptor corresponds to the translation product of the entire cDNA coding region. The receptor biochemically identified in the rabbit mammary gland is however much shorter. Thus, in the mammary gland, the receptor presumably undergoes post-translational modifications. The receptor forms L1, L2 and S bind prolactin with specificity and affinity similar to those reported for the native receptor. They also interact with two monoclonal antibodies, M110 and A917, specific for the native conformation of the hormone-binding site. The I1 and I2 forms do not bind prolactin, whereas the E form does. Thus, the hormone binding site is located in the extracellular domain which can function autonomously as a PRL-binding soluble protein. However, the E form binds prolactin with a higher affinity than the native receptor and it does not bind one of the two antireceptor monoclonal antibodies, known to be hormone binding-site specific. Thus, the conformation of the native receptor and that of the E form differ.
Cahoreau
C
C
Laboratoire de Pathologie Comparée, Institut National de la Recherche Agronomique, CNRS UA 1184, Saint-Christol-les-Alès, France.
Petridou
B
B
Cerutti
M
M
Djiane
J
J
Devauchelle
G
G
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D005822:Genetic Vectors; D013006:Growth Hormone / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D011388:Prolactin / Q000378:metabolism*; D011817:Rabbits; D011981:Receptors, Prolactin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection
D011981:Receptors, Prolactin; D011994:Recombinant Proteins; D011388:Prolactin; D013006:Growth Hormone; D004247:DNA