title: Purification and protein sequence analysis of rat liver prolactin receptor.
abstract: Prolactin receptors were purified from rat liver membranes by single-step immunoaffinity chromatography using a specific monoclonal antibody to the rat liver prolactin receptor. Scatchard analysis of 125I-human growth hormone binding to the purified receptor revealed two classes of specific binding sites with Ka = 18.5 x 10(9) and 1.2 x 10(9) M-1. Considering that both classes of binding sites are responsible for high affinity prolactin binding, the partially purified receptor preparation had a binding activity of 1.69 nmol/mg protein, representing 1000-fold purification over microsomal receptors with a recovery of 52%. From three separate purifications, 6 mg of partially purified prolactin receptor were obtained with a purity of approximately 4 to 6.5%. Thus, the use of monoclonal antibody for affinity chromatography resulted in a large improvement of prolactin receptor purification compared to previous hormone affinity chromatography (300-fold purification, 15% recovery). The purified receptor was run on preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a homogeneous preparation of prolactin receptor was obtained by electroelution from gel slices corresponding to Mr 38,000-43,000. Immunoblot analysis using a radiolabeled monoclonal antibody revealed two separate but closely located bands of Mr 42,000 and 40,000 in microsomal, partially purified, and electroeluted preparations. The homogeneous receptor protein was extensively digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone trypsin, and 10 internal amino acid sequences of the rat liver prolactin receptor were determined by gas-phase sequence analysis. Oligonucleotide probes were prepared against two of these internal sequences, and a prolactin receptor cDNA was isolated from a rat liver library using one of these probes (Boutin, J. M., Jolicoeur, C., Okamura, H., Gagnon, J., Edery, M., Shirota, M., Banville, D., Dusanter-Fourt, I., Djiane, J., and Kelly, P. A. (1988) Cell 53, 69-77). The amino acid sequence deduced from the cDNA reveals three potential sites of N-linked glycosylation, two of which were confirmed during protein sequencing. The prolactin receptor was characterized by affinity labeling with 125I-human growth hormone. Cross-linking of microsomes revealed a single band for the hormone-receptor complex with Mr 62,000. On the other hand, cross-linking of Triton X-100-solubilized or partially purified receptor with labeled hormone resulted in the appearance of two bands with Mr 62,000 and 102,000, suggesting the existence of a subunit structure of the prolactin receptor, or alternatively, the existence of two types of prolactin receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Laboratory of Molecular Endocrinology, McGill University, Royal Victoria Hospital, Montreal, Quebec, Canada.
authors.forename: H
authors.initials: H
authors.lastname: Okamura
authors.forename: S
authors.initials: S
authors.lastname: Raguet
authors.forename: A
authors.initials: A
authors.lastname: Bell
authors.forename: J
authors.initials: J
authors.lastname: Gagnon
authors.forename: P A
authors.initials: PA
authors.lastname: Kelly
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002846:Chromatography, Affinity; D005260:Female; D013006:Growth Hormone / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D011981:Receptors, Prolactin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D011981:Receptors, Prolactin; D013006:Growth Hormone