Biological activity and immunological reactivity of human prolactin mutants

We examined the biological activity and immunological reactivity of four mutants of human PRL. Two were mutants that changed the ability of human PRL to inhibit rat PRL storage when transfected into a rat pituitary cell line:mutations S34A and N31T. Two mutations were in regions of PRL that are highly conserved. One, des(3-11)-PRL, removed the N-terminal cystine loop that most PRLs, except those from certain fish, have, and no GHs have. The other, S90A, mutated a serine that is present in all PRLs but those from some fish and in all GHs. The immunological properties of des(3-11)-PRL were reduced 10-fold compared to those of wildtype human PRL in a RIA using NIH antihuman PRL-3, AFP C11580; the others were similar to those of wild-type PRL. The biological activity of des(3-11)-PRL was the most affected; activity was reduced about 8-fold compared to that of wild-type PRL in the Nb2 cell assay. The activities of the others were similar to that of the wild type. Serine 90 may be partially buried by loops connecting the alpha-helixes. The mutation of serine 90 did not affect the stability of the molecule in vitro, determined by comparing the red shift in tryptophan fluorescence that occurs with increasing concentrations of urea in S90A and wild-type PRL. The activity of S34A and N31T mutations indicates there is no correlation between biological activity and ability to affect storage. The N-terminal cystine loop may be conserved because it is needed for biological activity, but the conservation of serine 90 in GH and PRL must be determined by other properties, such as spacial requirements.