Production of recombinant rat proopiomelanocortin1-74 and characterization of its mitogenic action on pituitary lactotrophs.
We report the production of biologically active recombinant rat Gly-2-Ser-1-POMC1-74 (rrPOMC1-74) in a prokaryotic expression system. The polypeptide was produced as a fusion protein with glutathione-S-transferase (GST), using the pGEX-4T-1 vector and subsequently cleaved by thrombin. Amino acid sequencing, up to residue 45, showed a correct primary structure including the two additional amino acids at the N-terminus, Gly and Ser, derived from the thrombin cleavage site. Electrospray ionization mass spectrometry showed a Mr of 8358.5 Da which was 14-16 Da heavier (oxidation or methylation) than the calculated mass. Combined digestion with trypsin and endoproteinase Glu-C followed by MALDI-TOF mass spectrometry and N-terminal sequencing of the separated fragments showed a correct disulphide bridge configuration. In reaggregate cell cultures of immature rat pituitary, rrPOMC1-74 displayed biological activity similar to that of natural human (h) POMC1-76 or rat POMC1-74: it stimulated DNA replication in lactotrophs but not in other pituitary cell types. However, its efficacy was significantly lower than that of the natural product. Gamma3-MSH, a peptide that can be generated from POMC1-74 and a typical ligand of the melanocortin-3 (MC-3) receptor, also stimulated DNA replication in lactotrophs and, in contrast to rrPOMC1-74, also in somatotrophs and thyrotrophs. rrPOMC1-74 increased cAMP levels in 293HEK cells stably transfected with the MC-3 receptor with an intrinsic activity and potency similar to that of gamma3-MSH. However, natural hPOMC1-76 was inactive in the latter test system. These data show that rrPOMC1-74 mimics the selective mitogenic action of natural POMC1-74 on lactotrophs. Since natural POMC1-74 is N- and O-glycosylated and rrPOMC1-74 is not, glycosylation does not seem to determine the selectivity for lactotrophs. In spite of the feature that rrPOMC1-74 is an agonist at the MC-3 receptor and the reported evidence that the MC-3 receptor is expressed in the anterior pituitary, the mitogenic action of rrPOMC1-74 on lactotrophs does not seem to be mediated by the MC-3 receptor.
Bert
C
C
Laboratory of Cell Pharmacology, University of Leuven Medical School, Belgium.
Vande Vijver
V
V
Andries
M
M
Verhaert
P
P
Proost
P
P
De Vreese
B
B
Van Beeumen
J
J
Vankelecom
H
H
Denef
C
C
D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D005260:Female; D006133:Growth Substances / Q000494:pharmacology*; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D011333:Pro-Opiomelanocortin / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D011388:Prolactin / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018043:Receptors, Corticotropin / Q000378:metabolism; D044101:Receptors, Melanocortin; D011994:Recombinant Proteins / Q000096:biosynthesis*; D020539:Sequence Analysis, Protein; D013936:Thymidine / Q000378:metabolism; D014162:Transfection; D014316:Tritium
D006133:Growth Substances; D018043:Receptors, Corticotropin; D044101:Receptors, Melanocortin; D011994:Recombinant Proteins; D014316:Tritium; D011333:Pro-Opiomelanocortin; D011388:Prolactin; D000242:Cyclic AMP; D013936:Thymidine