Parathyroid hormone biosynthesis. Correlation of conversion of biosynthetic precursors with intracellular protein migration as determined by electron microscope autoradiography.

Abstract

The formation of parathyroid hormone (PTH) in the parathyroid gland occurs via two successive proteolytic cleavages from larger biosynthetic precursors. The initial product coded for by PTH mRNA is pre-proparathyroid hormone (PreProPTH), a polypeptide of 115 amino acids. Within 1 min of synthesis, the polypeptide, proparathyroid hormone (ProPTH), is formed as a result of the proteolytic removal of the NH2-terminal 25 amino acids from Pre-ProPTH. After a delay of 15-20 min, the NH2-terminal six-amino acid sequence of ProPTH is removed to give PTH of 84 amino acids. To investigate the subcellular sites in the parathyroid cell where the biosynthetic precursors undergo specific proteolytic cleavages, we examined, by electron microscope autoradiography, the spatiotemporal migration of autoradiographic grains and, by electrophoresis, the kinetics of the disappearance of labeled Pre-ProPTH and the conversion of labeled ProPTH to PTH in bovine parathyroid gland slices incubated with [3H]leucine for 5 min (pulse incubation) followed by incubations with unlabeled leucine for periods up to 85 min (chase incubations). By 5 rain, 85% of the autoradiographic grains were confined to the rough endoplasmic reticulum (RER). Autoradiographic grains increased rapidly in number in the Golgi region after 15 min of incubation; from 15 to 30 min they migrated within secretory vesicles still in the Golgi region and then migrated to mature secretory granules outside the Golgi area. Electrophoretic analyses showed that Pre-ProPTH disappeared rapidly (by 5 min) and that conversion of ProPTH to PTH was first detectable at 15 rain and was completed by 30 min. At later times of incubation (30-90 min), autoradiographic grains within the secretion granules migrated to the periphery of the cell and to the plasma membrane, in correlation with the release of PTH first detected by 30 min. We conclude that proteolytic conversion of Pre-ProPTH to ProPTH takes place in the RER and that subsequent conversion of ProPTH to PTH occurs in the Golgi complex. J. CELL BIOLOGY 9The Rockefeller University Press

The release of parathyroid hormone and the exocytosis of a proteoglycan are modulated by extracellular Ca2+ in a similar manner.

“…Because in bovine parathyroid the time necessary for the transport of PTH from the endoplasmic reticulum to Golgi is -20 min (Habener et al, 1979), the kinetics of transport of PTH and proteoglycan from the Golgi to plasma membrane appear comparable. Second, our results show that external Ca2+ inhibits the release of [35S]sulfated proteoglycan and immunoreactive PTH with a similar degree and in the same concentration range (Figure 7), suggesting the same mechanism of action on the secretion of these two products.…”

The Enzymology and Intracellular Organization of Peptide Precursor Processing: The Secretory Vesicle Hypothesis

“…Further experimental work supporting this view of the role of secretory vesicles in precursor processing can be found in various studies of proinsulin [21,22,47,80,91] and POMC [31,36,58,93] processing. In some cases (e.g., for parathyroid hormone processing [38], the interpretation is that precursor processing can occur in the Golgi complex. Such interpretations [31,[36][37][38]47,911] are usually based on kinetic analysis of pulse-chase data in combination with subcellular fractionation and/or radioautographic experi ments, and shold be viewed cautiously since it is very diffi cult to separate pure Golgi organelles from newly formed secretory vesicles by these methods.…”

“…Such interpretations [31,[36][37][38]47,911] are usually based on kinetic analysis of pulse-chase data in combination with subcellular fractionation and/or radioautographic experi ments, and shold be viewed cautiously since it is very diffi cult to separate pure Golgi organelles from newly formed secretory vesicles by these methods. Even in morphological analyses at the electron microscopic level, it is difficult to distinguish many trans-Golgi cisternae from immature se cretory vesicles [38]. Although possible roles for RER and Golgi in proteolytic processing of precursors cannot be eli minated.…”

Changes in cytoplasmic calcium determine the secretory response to extracellular cations in human parathyroid cells: a confocal microscopy study using FM1-43 dye

“…It was anticipated that such a cell model should be functional, because electron microscopy morphometry in rat parathyroid cells showed that a transient depression in [Ca# + ] ext induces initial centrifugal membrane shifting, indicating enhanced exocytosis, followed by centripetal membrane shifting, indicating endocytotic retrieval of plasma membrane [6,7].…”

“…Since in other endocrine cells secretion is stimulated rather than inhibited by an increase in [Ca# + ] i , this ' parathyroid paradox ' remains a challenging question. Furthermore, morphological studies reported that PTH is stored in cytoplasmic granules [5] found under the cytoplasmic membrane of cells from parathyroid adenomas [6] and which are lost during secretion induced by low [Ca# + ] ext [7]. These data suggest that, despite its inhibition by high [Ca# + ] i , PTH secretion occurs through regulated exocytosis.…”