Abstract
Unlike the neuroendocrine cell lines widely used to study trafficking of soluble and membrane proteins to secretory granules, the endocrine cells of the anterior pituitary are highly specialized for the production of mature secretory granules. Therefore, we investigated the trafficking of three membrane proteins in primary anterior pituitary endocrine cells. Peptidylglycine alpha-amidating monooxygenase (PAM), an integral membrane protein essential to the production of many bioactive peptides, is cleaved and enters the regulated secretory pathway even when expressed at levels 40-fold higher than endogenous levels. Myc-TMD/CD, a membrane protein lacking the lumenal, catalytic domains of PAM, is still stored in granules. Secretory granules are not the default pathway for all membrane proteins, because Tac accumulates on the surface of pituitary endocrine cells. Overexpression of PAM is accompanied by a diminution in its endoproteolytic cleavage and in its BaCl(2)-stimulated release from mature granules. Because internalized PAM/PAM-antibody complexes are returned to secretory granules, the endocytic machinery of the pituitary endocrine cells is not saturated. As in corticotrope tumor cells, expression of PAM or Myc-TMD/CD alters the organization of the actin cytoskeleton. PAM-mediated alterations in the cytoskeleton may limit maturation of PAM and storage in mature granules.
Highlights
Endocrine tissues, Peptidylglycine ␣-amidating monooxygenase (PAM) is one of a small number of posttranslational processing enzyme occurring naturally in soluble and membrane forms [1,2,3,4]
To elucidate the trafficking of membrane proteins in endocrine cells and understand the distinctly different steady state localization of endogenous PAM in primary pituitary cells and transfected PAM in AtT-20 cell lines, we investigated the trafficking of three membrane proteins in primary anterior pituitary: membrane PAM (PAM-1); a truncated version of PAM
Encoded PAM-1 is expressed at varying levels in essentially all of the primary pituitary cells (Fig. 1, A and B)
Summary
Endocrine tissues, PAM is one of a small number of posttranslational processing enzyme occurring naturally in soluble and membrane forms [1,2,3,4] For this reason, we have used PAM to investigate the trafficking of soluble and membrane proteins into secretory granules [5, 6]. As in AtT-20 cells, the endogenous PAM in pituitary cells is subjected to endoproteolytic cleavage, generating soluble PHM and PAL In both cases, PAM processing products are subjected to regulated exocytosis. To elucidate the trafficking of membrane proteins in endocrine cells and understand the distinctly different steady state localization of endogenous PAM in primary pituitary cells and transfected PAM in AtT-20 cell lines, we investigated the trafficking of three membrane proteins in primary anterior pituitary: membrane PAM (PAM-1); a truncated version of PAM. Overexpression of PAM alters the organization of the actin cytoskeleton, neither the regulated secretion or the endocytic machinery is affected in anterior pituitary cells
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