Prolactin proteolysis by glandular kallikrein: in vitro reaction requirements and cleavage sites, and detection of processed prolactin in vivo.
Glandular kallikrein (GK) is a trypsin-like serine protease characterized by an ability to generate bioactive peptides from inactive precursors with high specificity. In rat pituitary lactotrophs, GK is an estrogen-induced dopamine-repressed protein which is coregulated with PRL and is localized in organelles associated with precursor processing, suggesting a function to process PRL to novel forms with unique biological activities. To investigate this hypothesis we studied the ability of GK to cleave PRL in vitro and also examined rat pituitary extracts for processed forms of PRL using Western blot analysis. GK did not cleave rat PRL under routine incubation conditions. However, GK converted PRL from a 25K form to a 22K form (by gel electrophoresis) in the presence of thiols (dithiothreitol or mercaptoethanol) and Triton X-100. These reagents appear to elicit proteolysis by altering PRL's conformation; thiols were essential for proteolysis and Triton X-100 enhanced the thiol effects. Sequencing of processed PRL revealed 3 cleavage sites: Arg174-Arg175,Lys185-Phe186, and Arg188-Cys189. The cleavage sites are clustered close to the C-terminus on a 16-amino acid domain bracketed by cysteine residues. The cleavage sites in PRL resemble those of other GK substrates in their structural features and are in a highly conserved domain of PRL. Western blot analysis of rat pituitary extracts using antiserum against rat PRL revealed 22K PRL variants that were highly estrogen dependent and whose levels were markedly increased by in vivo administration of cysteamine, a biological thiol known to alter pituitary PRL conformation in vivo. The 22K PRL variants comigrated with PRL bands generated by sequential in vitro processing with GK and carboxypeptidase-B. The present findings support the hypothesis that GK may function to process PRL to novel forms in rat lactotrophs.
Powers
C A
CA
Department of Pharmacology, New York Medical College, Valhalla 10595.
Hatala
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007610:Kallikreins / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017830:Octoxynol; D010902:Pituitary Gland / Q000201:enzymology*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011388:Prolactin / Q000378:metabolism*; D051381:Rats; D020840:Tissue Kallikreins; D014357:Trypsin / Q000378:metabolism
D011092:Polyethylene Glycols; D011388:Prolactin; D017830:Octoxynol; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D007610:Kallikreins; D020840:Tissue Kallikreins; D014357:Trypsin; D004229:Dithiothreitol