Prolactin (PRL) Processing by Kallikrein: Production of the 21–23.5K PRL-Like Molecules and Inferences about PRL Storage in Mature Secretory Granules*

Abstract

We have previously described a series of C-terminally-clipped forms of PRL, the 21-23.5K PRL-like molecules (PLMs). Because we noted estrogen (E2) induction of PLMs and E2 also induces a pituitary glandular kallikrein, we have investigated the possibility that processing of PRL by kallikrein is responsible for the production of the PLMs. Subcellular fractionation of pituitaries from control or E2-treated female rats showed total kallikrein to be concentrated 1- to 4-fold and 6- to 20-fold in the granules (vs. original homogenate) from control and E2-treated animals, respectively. Cleavage of purified PRL by kallikrein resulted in the formation of large quantities of a number of the PLMs. Incubation of secretory granules derived from control or E2-treated animals under the same conditions showed no cleavage of PRL in the absence of a limiting granule membrane and a small production of the PLMs in the presence of a granule membrane. Production in the latter instance was slightly greater in granules derived from E2-treated animals. Addition of purified kallikrein to secretory granules from control or E2-treated animals in the presence or absence of granule membranes, resulted in the additional production of large amounts of only the smallest PLM (PLM 9), indicating a lack of cleavage of the two most C-terminal sites for the enzyme. Increasing the concentration of beta-mercaptoethanol to 0.64 M, to ensure monomerization of the granule PRL, had no effect on endogenous kallikrein activity of the number of products resulting from the addition of exogenous kallikrein to granules. In summary: 1) purified kallikrein can cleave purified PRL to form products which run with the same isoelectric point and mol wt values as the PLMs; 2) kallikrein is present in PRL secretory granules; 3) some PLM production occurs in a membrane-bound secretory granule fraction, but none occurs after removal of the membrane; this intragranular production, like cleavage of purified PRL with purified kallikrein, is dependent on the presence of a detergent and a reducing agent; 4) cleavage of granular PRL by exogenous kallikrein is limited to a single site and the more C-terminal sites are protected; and 5) protection of the C-terminus is not removed by intermolecular and intramolecular disulfide bond reduction. We conclude: 1) that pituitary glandular kallikrein is a strong candidate for the enzyme responsible for the production of the PLMs, and 2) that there is some element of PRL storage, other than intermolecular disulfide bonds, which involves the C-terminus of the molecule.

Secretion of Specific Nonphosphorylated and Phosphorylated Rat Prolactin Isoforms at Different Stages of the Estrous Cycle

“…Similarly, one can correlate rising estro gen levels first with an inhibition of the isoform 2 to 1 interconverting enzyme during diestrus 1 and 2 and later, at higher levels during proestrus, also with an inhibition of the isoform 3 to 3' interconverting enzyme. That estro gen treatment of intact animals might result in an inhibi tion of the activity of the 3 to 3' interconverting enzyme rather than its synthesis is suggested by results showing large amounts of isoform 3' plus some even more acidic isoforms is isolated activated mature granules (originally containing only isoform 2) from estrogen-treated animals [25], Thus plenty of enzyme is present in the granules which must be normally tonically inhibited in the intact cell. The normal tonic inhibition of most of the converting enzyme activity in the intact pituitary probably accounts for the lack of correlation between intracellular and secreted PRL.…”

Identification of the Major Site of Rat Prolactin Phosphorylation as Serine 177

“…Under these conditions about 30% of the PRL is phosphorylated, as assessed by densitometric analysis of the phosphoprotein on two-dimensional protein gels. Besides PRL, which is a major phosphoprotein, a phosphoprotein at approximately 21 kDa, which is most likely a previously characterized cleaved fragment of PRL produced by the granular kallikrein (17), is seen (see later for further analysis). This 21-kDa protein is produced in granules which have been incubated at 37°C and hence it is not present in unincubated granules such as those illustrated in Fig.…”

“…In a series of granule preparations, PRL constituted between 94 and 97% of total protein. A number of other proteins are present in small quantities some of which must be the kinases (12), a protease (17), and a disulfide isomerase (18) previously described as granule constituents. PRL was identified by Western blot analysis of a duplicate to lane 1 (lane 2) and by approximate molecular mass as compared to standards (lane 3).…”

“…Following intragranular phosphorylation by endogenous kinases, digestion with exogenous kallikrein was initiated. Kallikrein is a natural component of PRL secretory granules (17) and so some cleavage was expected in the samples to which exogenous kallikrein was not added as soon as the requirement for ␤-mercaptoethanol and Triton X-100 was fulfilled in the experimental protocol (necessary for efficient kallikrein cleavage of PRL) (19). Electrophoresis and autoradiographic analysis of this control (Fig.…”

Characterization of Cysteamine Induction of the 22K Prolactin Variant in the Rat Pituitary

“…The 21K PRL variant could not be attributed to lysosomal degradation, and appeared to arise from C-terminal cleavage -consistent with GK processing. More recent studies have shown that GK cleavage products comigrate with the 21K PRL variant during 2-D gel electrophoresis [19]. It is also of note that Liu et al [20] reported a 21K PRL variant in human serum, suggesting the secretion of a GK-processed PRL in man.…”