Kalirin, a Cytosolic Protein with Spectrin-like and GDP/GTP Exchange Factor-like Domains That Interacts with Peptidylglycine α-Amidating Monooxygenase, an Integral Membrane Peptide-processing Enzyme

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 ...

An Isoform of Kalirin, a Brain-specific GDP/GTP Exchange Factor, Is Enriched in the Postsynaptic Density Fraction

“…Moreover, Kalirin-8 was previously shown to bind nucleotide-depleted Rac1, but not nucleotide-depleted RhoA or Cdc42 (10). Hence, we tested whether the DH-PH domain of Kalirin could activate in vitro the release of bound GDP from Rac1 (Fig.…”

“…Several Dbl family members, including Ost (11) and Tiam (12), are expressed in the brain, but only Kalirin is restricted to the central nervous system. AtT-20 corticotrope tumor cells overexpressing one form of Kalirin (Kalirin-8) and peptidylglycine-␣-amidating monooxygenase developed longer and more branched neuritic processes (10). Both AtT-20 and Chinese hamster ovary cells overexpressing Kalirin-8 displayed rearrangements of the actin cytoskeleton (13).…”

Plasticity in Hippocampal Peptidergic Systems Induced by Repeated Electroconvulsive Shock

“…Kalirin, identified by its ability to interact with routing determinants in the cytosolic domain of PAM, is a GDP/GTP exchange factor for selected Rho family small GTP binding proteins (Alam et al 1997). Rho proteins mediate cytoskeletal reorganization and play a central role in the specification of dendritic morphology (Threadgill et al 1997;Hall 1998).…”

Trio : Un facteur d’échange des GTPases Rho aux multiples facettes impliqué dans le guidage axonal

“…Une hypothèse possible pour expliquer le faible effet de l'absence de Trio sur le développement cérébral est l'existence d'une redondance fonctionnelle avec des protéines de la même famille. En effet, chez les mammifères, la kalirine chez le rat et son orthologue Duo/Duet chez l'homme ont une organisation structurale très similaire à celle de Trio [30][31][32] (figure 2). L'expression de la kalirine est restreinte au cerveau adulte, et une étude récente a montré qu'elle serait impliquée dans le remodelage du cytosquelette d'actine dans les dendrites de neurones corticaux, réglant ainsi leur morphologie [33].…”