No abstract

Although the integral membrane proteins that catalyze steps in the biosynthesis of neuroendocrine peptides are known to contain routing information in their cytosolic domains, the proteins recognizing this routing information are not known. Using the yeast two-hybrid system, we previously identified P-CIP10 as a protein interacting with the cytosolic routing determinants of peptidylglycine ␣-amidating monooxygenase (PAM). P-CIP10 is a 217-kDa cytosolic protein with nine spectrinlike repeats and adjacent Dbl homology and pleckstrin homology domains typical of GDP/GTP exchange factors. In the adult rat, expression of P-CIP10 is most prevalent in the brain. Corticotrope tumor cells stably expressing P-CIP10 and PAM produce longer and more highly branched neuritic processes than nontransfected cells or cells expressing only PAM. The turnover of newly synthesized PAM is accelerated in cells co-expressing P-CIP10. P-CIP10 binds to selected members of the Rho subfamily of small GTP binding proteins (Rac1, but not RhoA or Cdc42). P-CIP10 (kalirin), a member of the Dbl family of proteins, may serve as part of a signal transduction system linking the catalytic domains of PAM in the lumen of the secretory pathway to cytosolic factors regulating the cytoskeleton and signal transduction pathways.Cytosolic proteins are involved in the formation of secretory granules (1-6) and in the trafficking and localization of integral membrane proteins needed for the synthesis of bioactive peptides (7-16). We have used one of the few integral membrane proteins known to be involved in the biosynthesis of neuropeptides, peptidylglycine ␣-amidating monooxygenase (PAM), 1 to search for cytosolic proteins involved in these processes (17). PAM is a bifunctional enzyme and integral membrane forms contain an NH 2 -terminal signal sequence, the two catalytic domains that catalyze the sequential reactions required for peptide amidation, a single transmembrane domain, and a short cytosolic domain (18). PAM is involved in the production of all ␣-amidated peptides and functions only after neuroendocrine-specific endoproteases and carboxypeptidases have exposed the COOH-terminal glycine residue that serves as the nitrogen donor for amide formation (19). Immunocytochemical evidence indicates that PAM begins to function in the trans-Golgi network (TGN), but most peptide amidation occurs in immature secretory granules (20). Using immunoelectron microscopy, integral membrane forms of PAM have been localized to the TGN, especially to distal tubuloreticular regions, and to large dense core vesicles (21).When expressed independently in neuroendocrine cells, each lumenal catalytic domain of PAM is targeted to large dense core vesicles (22). Integral membrane forms of PAM are localized to the TGN region of both neuroendocrine and nonneuroendocrine cells (7,8). A small percentage of membrane PAM is present on the cell surface or in endosomes at steady state. Elimination of the distal 40 amino acids of the 86-amino acid cytosolic domain results in relocation of ...

The steady state distribution of membrane forms of peptidylglycine ␣-amidating monooxygenase (PAM) in the secretory pathway of neurons and endocrine cells depends on signals in its cytosolic COOH-terminal domain (CD). Mutagenesis studies yielded catalytically active PAM proteins that are not properly localized or internalized. Employing the yeast two-hybrid system, we isolated two distinct cDNAs whose protein products showed a strong interaction with the CD of PAM. The interaction of these novel PAM COOH-terminal interactor proteins (P-CIPs) did not occur with a misrouted CD mutant as bait in the yeast system. Both proteins, P-CIP2 and P-CIP10, were expressed as fusion proteins that interacted in vitro with solubilized integral membrane PAM. P-CIP2 was homologous to several serine/ threonine and dual specificity protein kinases, while P-CIP10 contained spectrin-like repeats. Endogenous P-CIP2 was localized to the Golgi region of AtT-20 corticotrope tumor cells, and expression of integral membrane PAM disrupted the distribution of endogenous P-CIP2. Both P-CIP2 and P-CIP10 mRNAs were found to be expressed in rat brain neurons also expressing PAM proteins. P-CIP2 and P-CIP10 may be members of a family of cytosolic proteins involved in the routing of membrane proteins that function in the regulated secretory pathway.