No abstract

Two related proteins, inhibin and activin, are produced and secreted by the gonads and act at the pituitary to regulate FSH secretion. In the present study, the alpha and beta B, but not the beta A, polypeptide subunits of inhibin were localized in the cytoplasm of FSH- and LH-immunoreactive (ir) gonadotropes. Ovariectomy (OVX) increased the size and number of cells immunoreactive for inhibin-alpha and -beta B as well as the mRNAs encoding these subunits. Treatment with estrogen prevented these effects. These results suggest that pituitary gonadotropes are sources, as well as targets, of inhibin-related peptides, whose expression in the pituitary is modulated by ovarian factors.

Two series of CRF antagonists with N alpha- and C alpha-methylated alanine and leucines were evaluated for their biological activities in vitro and in vivo in several systems. The poly-N-methylated analogue of alpha-helical-CRF9-41, [N alpha MeLeu10,15,27,37,N alpha MeAla22,32,41]-alpha-Hel-CRF9-41, was found to be considerably less potent than the parent non-N-methylated analogue. This result was expected on the basis that alpha-helicity was thought to be required for biological activity and the prediction that backbone substitutions on the nitrogen have a tendency to break alpha-helices (a hypothesis that was confirmed by circular dichroism). Next, a series of constrained analogues of the potent CRF antagonist, [DPhe12,Nle21,38]h/rCRF12-41, was synthesized that contained C alpha-methylleucine and/or C alpha-methylalanine (Aib) residues at selected positions. Because C alpha-methylation is recognized to increase alpha-helicity, and because there is now strong NMR data suggesting that residues 6-36 assume a well-defined alpha-helix, it was expected that these analogues would be more potent. Although usual solid-phase peptide synthesis procedures were followed, success in coupling the C alpha-methyl amino acids was obtained only with a 1:1 mixture of BOP/HOBt. In vitro potencies of the synthesized compounds were measured in a collagenase-dispersed anterior pituitary cell culture bioassay. Monosubstituted analogues were shown to be twice to one fourth as potent as the parent compound; while the pluri-substituted peptides were slightly less potent. This decrease in potency might be correlated to an unexpected lower helical content of the pluri-substituted compounds (as determined by CD spectroscopy), as it was suggested that the bioactive conformation of the CRF was predominantly alpha-helical. Interestingly, one analogue, [DPhe12,Nle21,38,C alpha-MeLeu37]h/rCRF12-41, was found to be more potent and longer acting than the parent compound in two in vivo assays measuring ACTH release after intravenous administration to adrenalectomized rats and reversal of stress-induced delay in gastric emptying in the rat after intracisternal administration. The molecular basis for this increased duration of action and potency is being investigated.

The binding of a ligand to cell surface receptors initiates a cascade of intracellular signals that generate responses to the external stimuli. Thus, this event plays a pivotal role in the mechanism of transmembrane signaling. Activin is a member of a cytokine family that is involved in diverse biological processes. To study the structural basis that underlies the transmembrane signaling mechanism, we have overexpressed the soluble extracellular domain of the type II activin receptor from mouse (ActRII-ECD). We used the methylotrophic yeast Pichia pastoris as an expression host to produce a large quantity of ActRII-ECD. Expression was carried out in a fermentor with a typical yield of 10 mg of pure ActRII-ECD from a liter of growth media. Biological function was confirmed by the ability to decrease the activin-stimulated release of FSH from cultured rat pituitary cells in addition to several activin-binding assays, including native gel shift and chemical cross-linking. The glycosylation on ActRII-ECD was shown to be dispensable for high-affinity activin binding, and nonnatural sugars from the yeast expression host did not interfere with binding, indicating that the binding of activin is not sensitive to the environment near the two positions of N-linked glycosylation. Analytical ultracentrifugation of the complex between activin A and ActRII-ECD reveals that two receptors associate with one activin A dimer, consistent with results from chemical cross-linking experiments.