Production of recombinant rat proopiomelanocortin1–74 and characterization of its mitogenic action on pituitary lactotrophs

Abstract

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 M r of 8358.5 Da which was 14–16 Da heavier (oxydation 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. γ3-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 γ3-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.