Biomedical subjects

B A Eipper

Publications and source records attributed to B A Eipper.

At least 37 records · Page 2Linked to original sources

PubMed2000

Cell type-specific storage of dopamine beta-monooxygenase.

Expression of dopamine beta-monooxygenase (DBM), the enzyme that converts dopamine into norepinephrine, is limited to adrenal chromaffin cells and a small population of neurons. We studied DBM trafficking to regulated granules by stably expressing rat DBM in AtT-20 corticotrope tumor cells, which contain regulated granules, and in Chinese hamster ovary (CHO) cells, which lack regulated granules. The behavior of exogenous DBM in both cell lines was compared with endogenous DBM in adrenal chromaffin cells. CHO cells secreted active DBM, indicating that production of active enzyme does not require features unique to neuroendocrine cells. Pulse-chase experiments indicated that early steps in DBM maturation followed a similar time course in AtT-20, CHO, and adrenal chromaffin cells. Use of a conformation-sensitive DBM antiserum indicated that acquisition of a folded structure occurred with a similar time course in all three cell types. Cell type-specific differences in DBM trafficking became apparent only when storage in granules was examined. As expected, DBM was stored in secretory granules in chromaffin cells; CHO cells failed to store DBM. Despite the fact that AtT-20 cells have regulated granules, exogenous DBM was not stored in these granules. Thus storage of DBM in secretory granules requires cell type specific factors.

Animals↗

PubMed2000

New insights into copper monooxygenases and peptide amidation: structure, mechanism and function.

Many bioactive peptides must be amidated at their carboxy terminus to exhibit full activity. Surprisingly, the amides are not generated by a transamidation reaction. Instead, the hormones are synthesized from glycine-extended intermediates that are transformed into active amidated hormones by oxidative cleavage of the glycine N-C alpha bond. In higher organisms, this reaction is catalyzed by a single bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The PAM gene encodes one polypeptide with two enzymes that catalyze the two sequential reactions required for amidation. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) catalyzes the stereospecific hydroxylation of the glycine alpha-carbon of all the peptidylglycine substrates. The second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5), generates alpha-amidated peptide product and glyoxylate. PHM contains two redox-active copper atoms that, after reduction by ascorbate, catalyze the reduction of molecular oxygen for the hydroxylation of glycine-extended substrates. The structure of the catalytic core of rat PHM at atomic resolution provides a framework for understanding the broad substrate specificity of PHM, identifying residues critical for PHM activity, and proposing mechanisms for the chemical and electron-transfer steps in catalysis. Since PHM is homologous in sequence and mechanism to dopamine beta-monooxygenase (DBM; EC 1.14.17.1), the enzyme that converts dopamine to norepinephrine during catecholamine biosynthesis, these structural and mechanistic insights are extended to DBM.

Amidine-Lyases↗

PubMed2000

Breeding stock-specific variation in peptidylglycine alpha-amidating monooxygenase messenger ribonucleic acid splicing in rat pituitary.

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the carboxyl-terminal amidation of glycine-extended peptides in a two-step reaction involving a monooxygenase and a lyase. Several forms of PAM messenger RNA result from alternative splicing of the single copy PAM gene. The presence of alternately spliced exon A between the two enzymatic domains allows endoproteolytic cleavage to occur in selected tissues, generating soluble monooxygenase and membrane lyase from integral membrane PAM. While using an exon A antiserum, we made the unexpected observation that Charles River Sprague Dawley rats expressed forms of PAM containing exon A in their pituitaries, whereas Harlan Sprague Dawley rats did not. Forms of PAM containing exon A were expressed in the atrium and hypothalamus of both types of Sprague Dawley rat, although in different proportions. PAM transmembrane domain splicing also differed between rat breeders, and full-length PAM-1 was not prevalent in the anterior pituitary of either type of rat. Despite striking differences in PAM splicing, no differences in levels of monooxygenase or lyase activity were observed in tissue or serum samples. The splicing patterns of other alternatively spliced genes, pituitary adenylate cyclase-activating polypeptide receptor type 1 and cardiac troponin T, did not vary with rat breeder. Strain-specific variations in the splicing of transcripts such as PAM must be taken into account in analyzing the resultant proteins, and knowledge of these differences should identify variations with functional significance.

Alternative Splicing↗

PubMed2000

Cell type-specific metabolism of peptidylglycine alpha-amidating monooxygenase in anterior pituitary.

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme expressed in each major anterior pituitary cell type. We used primary cultures of adult male rat anterior pituitary to examine PAM expression, processing, and secretion in the different pituitary cell types and to compare these patterns to those observed in transfected AtT-20 corticotrope tumor cells. Immunostaining and subcellular fractionation identified PAM in pituitary secretory granules and additional vesicular compartments; in contrast, in AtT-20 cells, transfected PAM was primarily localized to the trans-Golgi network. PAM expression was highest in gonadotropes, with moderate levels in somatotropes and thyrotropes and lower levels in corticotropes and lactotropes. Under basal conditions, less than 1% of the cell content of monooxygenase activity was secreted per h, a rate comparable to the basal rate of release of individual pituitary hormones. General secretagogues stimulated PAM secretion 3- to 5-fold. Stimulation with specific hypothalamic releasing hormones demonstrated that different pituitary cell types secrete characteristic sets of PAM proteins. Gonadotropes and thyrotropes release primarily monofunctional monooxygenase. Somatotropes secrete primarily bifunctional PAM, whereas corticotropes secrete a mixture of mono- and bifunctional proteins. As observed in transfected AtT-20 cells, pituitary cells rapidly internalize the PAM/PAM-antibody complex from the cell surface. The distinctly different steady-state localizations of endogenous PAM in primary pituitary cells and transfected PAM in AtT-20 cell lines may simply reflect the increased storage capacity of primary pituitary cells.

Adrenocorticotropic Hormone↗

PubMed1999

The novel kinase peptidylglycine alpha-amidating monooxygenase cytosolic interactor protein 2 interacts with the cytosolic routing determinants of the peptide processing enzyme peptidylglycine alpha-amidating monooxygenase.

The cytosolic domain of the peptide-processing integral membrane protein peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14. 17.3) contains multiple signals determining its subcellular localization. Three PAM cytosolic interactor proteins (P-CIPs) were identified using the yeast two hybrid system (Alam, M. R., Caldwel, B. D., Johnson, R. C., Darlington, D. N., Mains, R. E., and Eipper, B. A. (1996) J. Biol. Chem. 271, 28636-28640); the partial amino acid sequence of P-CIP2 suggested that it was a protein kinase. In situ hybridization and immunocytochemistry show that P-CIP2 is expressed widely throughout the brain; PAM and P-CIP2 are expressed in the same neurons. Based on subcellular fractionation, the 47-kDa P-CIP2 protein is mostly cytosolic. P-CIP2 is a highly selective kinase, phosphorylating the cytosolic domain of PAM, but not the corresponding region of furin or carboxypeptidase D. Although P-CIP2 interacts with stathmin, it does not phosphorylate stathmin. Site-directed mutagenesis, phosphoamino acid analysis, and use of synthetic peptides demonstrate that PAM-Ser(949) is the major site phosphorylated by P-CIP2. Based on both in vitro binding experiments and co-immunoprecipitation from cell extracts, P-CIP2 interacts with PAM proteins containing the wild type cytosolic domain, but not with mutant forms of PAM whose trafficking is disrupted. P-CIP2, through its highly selective phosphorylation of a key site in the cytosolic domain of PAM, appears to play a critical role in the trafficking of this protein.

Amino Acid Sequence↗

PubMed1999

Phosphorylation of cytosolic domain Ser(937) affects both biosynthetic and endocytic trafficking of peptidylglycine alpha-amidating monooxygenase.

Peptidylglycine alpha-amidating monooxygenase (PAM), a bifunctional enzyme, catalyzes the COOH-terminal amidation of bioactive peptides. In test tube assays, PAM is phosphorylated by protein kinase C at Ser(937). The roles of phosphorylation and dephosphorylation of Ser(937) in the biosynthetic and endocytic trafficking of integral membrane PAM were examined using an antiserum specific for the phosphorylation of Ser(937) and using AtT-20 cells expressing membrane PAM in which Ser(937) was mutated to Ala or Asp. Although phosphorylation at Ser(937) can occur while PAM is in the endoplasmic reticulum, early steps in the biosynthetic trafficking of membrane PAM were not affected by Ser(937) phosphorylation. The inability to phosphorylate PAM/S937A increased its intracellular degradation and decreased secretion of the soluble monooxygenase portion of PAM. In contrast, the biosynthetic trafficking of PAM/S937D was indistinguishable from wild-type PAM. Despite the fact that Ser(937) is adjacent to the only Tyr-based internalization motif in PAM, internalization and trafficking through early endosomes were unaffected by phosphorylation. However, PAM antibody internalized by wild-type PAM acquired a perinuclear localization, while antibody internalized by PAM/S937A was routed to lysosomes, and antibody bound to PAM/S937D maintained a dispersed, punctate pattern. In cells stimulated with phorbol ester, phosphorylation of Ser(937) increased and phosphorylated PAM accumulated in large vesicular structures. Therefore, phosphorylation of PAM-1 at Ser(937) directs newly synthesized and internalized protein away from lysosomes, while dephosphorylation is needed for a different step in the late endocytic pathway.

Animals↗

PubMed1999

Induction of integral membrane PAM expression in AtT-20 cells alters the storage and trafficking of POMC and PC1.

Peptidylglycine alpha-amidating monooxygenase (PAM) is an essential enzyme that catalyzes the COOH-terminal amidation of many neuroendocrine peptides. The bifunctional PAM protein contains an NH2-terminal monooxygenase (PHM) domain followed by a lyase (PAL) domain and a transmembrane domain. The cytosolic tail of PAM interacts with proteins that can affect cytoskeletal organization. A reverse tetracycline-regulated inducible expression system was used to construct an AtT-20 corticotrope cell line capable of inducible PAM-1 expression. Upon induction, cells displayed a time- and dose-dependent increase in enzyme activity, PAM mRNA, and protein. Induction of increased PAM-1 expression produced graded changes in PAM-1 metabolism. Increased expression of PAM-1 also caused decreased immunofluorescent staining for ACTH, a product of proopiomelanocortin (POMC), and prohormone convertase 1 (PC1) in granules at the tips of processes. Expression of PAM-1 resulted in decreased ACTH and PHM secretion in response to secretagogue stimulation, and decreased cleavage of PC1, POMC, and PAM. Increased expression of a soluble form of PAM did not alter POMC and PC1 localization and metabolism. Using the inducible cell line model, we show that expression of integral membrane PAM alters the organization of the actin cytoskeleton. Altered cytoskeletal organization may then influence the trafficking and cleavage of lumenal proteins and eliminate the ability of AtT-20 cells to secrete ACTH in response to a secretagogue.

Actins↗

PubMed1999

Kalirin, a multifunctional PAM COOH-terminal domain interactor protein, affects cytoskeletal organization and ACTH secretion from AtT-20 cells.

The production and regulated secretion of bioactive peptides require a series of lumenal enzymes to convert inactive precursors into bioactive peptides plus several cytosolic proteins to govern granule formation, maturation, translocation, and exocytosis. Peptidylglycine alpha-amidating monooxygenase (PAM), an enzyme essential for biosynthesis of many peptides, is an integral membrane protein with trafficking information in both its lumenal and cytosolic domains. Kalirin, a PAM cytosolic domain interactor protein with spectrin-like repeats and GDP/GTP exchange factor activity for Rac1, is expressed with PAM in neurons but is not expressed in the anterior pituitary or AtT-20 corticotrope cells. Expression of Kalirin alters the cytoskeletal organization of Chinese hamster ovary and AtT-20 cells expressing membrane PAM. Expression of membrane PAM also alters cytoskeletal organization, demonstrating the presence of endogenous proteins that can mediate this effect. Significant amounts of both PAM and Kalirin fractionate with cytoskeletal elements. Since cytoskeletal organization is critical for exocytosis, constitutive-like and regulated secretions were evaluated. Whereas the constitutive-like secretion of adrenocorticotropic hormone (ACTH) is increased by expression of membrane PAM, regulated secretion is eliminated. Expression of Kalirin in AtT-20 cells expressing membrane PAM restores stimulated secretion of ACTH. Thus, Kalirin or its homologue may be essential for regulated secretion, and the PAM-Kalirin interaction may coordinate intragranular with cytosolic events.

Actins↗

PubMed1999

Kalirin inhibition of inducible nitric-oxide synthase.

Nitric oxide (NO) acts as a neurotransmitter. However, excess NO produced from neuronal NO synthase (nNOS) or inducible NOS (iNOS) during inflammation of the central nervous system can be neurotoxic, disrupting neurotransmitter and hormone production and killing neurons. A screen of a hippocampal cDNA library showed that a unique region of the iNOS protein interacts with Kalirin, previously identified as an interactor with a secretory granule peptide biosynthetic enzyme. Kalirin associates with iNOS in vitro and in vivo and inhibits iNOS activity by preventing the formation of iNOS homodimers. Expression of exogenous Kalirin in pituitary cells dramatically reduces iNOS inhibition of ACTH secretion. Thus Kalirin may play a neuroprotective role during inflammation of the central nervous system by inhibiting iNOS activity.

Adrenocorticotropic Hormone↗

PubMed1999

Substrate-mediated electron transfer in peptidylglycine alpha-hydroxylating monooxygenase.

Peptide amidation is a ubiquitous posttranslational modification of bioactive peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first step of this reaction, is composed of two domains, each of which binds one copper atom. The coppers are held 11 A apart on either side of a solvent-filled interdomain cleft, and the PHM reaction requires electron transfer between these sites. A plausible mechanism for electron transfer might involve interdomain motion to decrease the distance between the copper atoms. Our experiments show that PHM catalytic core (PHMcc) is enzymatically active in the crystal phase, where interdomain motion is not possible. Instead, structures of two states relevant to catalysis indicate that water, substrate and active site residues may provide an electron transfer pathway that exists only during the PHM catalytic cycle.

Animals↗

PubMed1999

A molluscan peptide alpha-amidating enzyme precursor that generates five distinct enzymes.

Mechanisms underlying the specificity and efficiency of enzymes, which modify peptide messengers, especially with the variable requirements of synthesis in the neuronal secretory pathway, are poorly understood. Here, we examine the process of peptide alpha-amidation in individually identifiable Lymnaea neurons that synthesize multiple proproteins, yielding complex mixtures of structurally diverse peptide substrates. The alpha-amidation of these peptide substrates is efficiently controlled by a multifunctional Lymnaea peptidyl glycine alpha-amidating monooxygenase (LPAM), which contains four different copies of the rate-limiting Lymnaea peptidyl glycine alpha-hydroxylating monooxygenase (LPHM) and a single Lymnaea peptidyl alpha-hydroxyglycine alpha-amidating lyase. Endogenously, this zymogen is converted to yield a mixture of monofunctional isoenzymes. In vitro, each LPHM displays a unique combination of substrate affinity and reaction velocity, depending on the penultimate residue of the substrate. This suggests that the different isoenzymes are generated in order to efficiently amidate the many peptide substrates that are present in molluscan neurons. The cellular expression of the LPAM gene is restricted to neurons that synthesize amidated peptides, which underscores the critical importance of regulation of peptide alpha-amidation.

Amino Acid Sequence↗

PubMed1999

Differential regulation of peptide alpha-amidation by dexamethasone and disulfiram.

alpha-Amidation is essential for the function of many peptides in intercellular communication. This C-terminal modification is mediated in a two-step process by the hydroxylase and lyase activities of the bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The first step, catalyzed by peptidylglycine-alpha-hydroxylating monooxygenase (PHM; EC 1.14.17. 3), is rate limiting in the process, and therefore subject to regulation. Dexamethasone and disulfiram (tetraethylthiuram disulfide; Antabuse) were used as in vivo treatments to study the regulation of PHM expression and activity in cardiac atrium. Our findings show that both dexamethasone and disulfiram treatment increase the activity of PHM in atrial tissue but that they do so by distinctly different mechanisms. Dexamethasone elevated tissue levels of PAM mRNA and protein concurrently, suggesting that glucocorticoids regulate PAM expression at the level of gene transcription. In contrast, disulfiram treatment, which depletes stores of alpha-amidated peptides, increased the specific activity of PHM without affecting the level of PAM expression. The catalytic efficiency of PHM was enhanced by raising the Vmax of the enzyme. Importantly, this increase in Vmax was retained through purification to homogeneity, indicating that either a covalent modification or a stable conformational change had occurred in the protein. These novel findings demonstrate that the rate-limiting enzyme in the bioactivation of peptide messengers is differentially regulated by transcriptional and post-transcriptional mechanisms in vivo. It is proposed that regulation of PHM's expression and catalytic efficiency serve as coordinated physiologic mechanisms for maintaining appropriate levels of alpha-amidating activity under changing conditions in vivo.

Animals↗

PubMed1999

In vivo inhibition of peptidylglycine-alpha-hydroxylating monooxygenase by 4-phenyl-3-butenoic acid.

Peptidylglycine-alpha-hydroxylating monooxygenase (PHM; EC 1.14.17. 3) catalyzes the first and rate-limiting reaction in the two-step process that alpha-amidates neural and endocrine peptides. The substrate analog 4-phenyl-3-butenoic acid (PBA) was shown in vitro to selectively inhibit PHM without affecting the activity of peptidyl-alpha-hydroxyglycine alpha-amidating lyase, the enzyme that mediates the second reaction in alpha-amidation. Inhibition of PHM activity by PBA lowered the V(max) of the enzyme without altering its K(m). Administration of PBA in vivo profoundly inhibited serum PHM activity in a dose- and time-related fashion. Maximal reductions to less than 5% of control levels were observed 3 h after a single administration (500 mg/kg). Inhibition of serum PHM activity by PBA was short-lived, being fully reversed by 24 h postinjection. PHM activity in cardiac atrium, hypothalamus, and anterior and neurointermediate lobes of the pituitary were also decreased by PBA treatment but to a lesser extent than with serum. Inhibition of PHM activity by PBA was not cumulative over time when assessed 24 h after the last of 10 daily injections (500 mg/kg). The role of protein synthesis in maintaining PHM activity in blood was demonstrated by treatment with cycloheximide, which reduced serum PHM activity and retarded the recovery of PHM activity after PBA administration. It is concluded that the metabolism and/or clearance of PBA is rapid and that de novo protein synthesis has an important role in mediating the rapid restoration of PHM activity after PBA administration.

Animals↗

PubMed1998

Post-translational N-glycosylation of a truncated form of a peptide processing enzyme.

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the carboxyl-terminal amidation of bioactive peptides through a two-step reaction involving the monooxygenase and lyase domains. PAM undergoes endoproteolytic cleavage in neuroendocrine cells in the lyase domain. To determine which of the two possible paired basic sites is utilized, truncated PAM proteins ending at these sites were stably expressed in Chinese hamster ovary cells. While characterizing the truncation mutants, it became apparent that N-glycosylation occurred post-translationally at the single site localized near the carboxyl terminus of the lyase domain. The post-translational N-glycosylation of this site does not require the newly synthesized protein to remain tightly bound to membranes. Both malfolded, secretion incompetent proteins and fully active, secreted proteins were subject to post-translational N-glycosylation.

Animals↗

PubMed1997

Embryonic and uterine expression patterns of peptidylglycine alpha-amidating monooxygenase transcripts suggest a widespread role for amidated peptides in development.

Posttranslational processing of peptide precursors frequently includes COOH-terminal amidation by the bifunctional enzyme peptidylglycine alpha-amidating monooxygenase (PAM). We examined the ontogeny of PAM gene expression using in situ hybridization and detected expression in the cardiogenic region beginning at embryonic day 9 (e9) and in decidualizing uterine endometrium and myometrial smooth muscle at even earlier postimplantation stages. PAM expression in the CNS at e10 was highest in the dorsal spinal cord and floor plate and exhibited complex patterning in several CNS regions, including the ventricular zone, over the next several days with PAM expression first detected in neurons at e13. High levels of PAM expression characterized several nonneural cell populations as well, including limb mesoderm and the mesenchyme immediately adjacent to nasal, maxillary, palatal, and dental epithelia during tissue fusion and remodeling. Since alternative splicing generates PAM transcripts encoding proproteins that are differentially localized and processed, we used probes that distinguish major subsets of PAM transcripts to determine that transcripts encoding integral membrane PAM isoforms predominate in most, if not all, PAM-expressing cell types throughout development. Further, transcripts that encode soluble and cleavable PAM isoforms are essentially absent from two CNS areas that are rich in transcripts encoding integral membrane, bifunctional PAM: the ependymal region of the spinal cord and the ventricular zone of the hippocampus. These results provide evidence for widespread expression and cell-type-specific alternative splicing of PAM during development and raise the possibility that region-specific amidation of PAM substrates contributes significantly to several developmental processes.

Animals↗

PubMed1997

Amidation of bioactive peptides: the structure of peptidylglycine alpha-hydroxylating monooxygenase.

Many neuropeptides and peptide hormones require amidation at the carboxyl terminus for activity. Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the amidation of these diverse physiological regulators. The amino-terminal domain of the bifunctional PAM protein is a peptidylglycine alpha-hydroxylating monooxygenase (PHM) with two coppers that cycle through cupric and cuprous oxidation states. The anomalous signal of the endogenous coppers was used to determine the structure of the catalytic core of oxidized rat PHM with and without bound peptide substrate. These structures strongly suggest that the PHM reaction proceeds via activation of substrate by a copper-bound oxygen species. The mechanistic and structural insight gained from the PHM structures can be directly extended to dopamine beta-monooxygenase.

Animals↗

PubMed1997

Peptidylglycine alpha-hydroxylating monooxygenase: active site residues, disulfide linkages, and a two-domain model of the catalytic core.

Peptidylglycine alpha-hydroxylating monooxygenase (PHM) is a copper, ascorbate, and molecular oxygen dependent enzyme that catalyzes the first step leading to the C-terminal amidation of glycine-extended peptides. The catalytic core of PHM (PHMcc), refined to residues 42-356 of the PHM protein, was expressed at high levels in CHO (DG44) (dhfr-) cells. PHMcc has 10 cysteine residues involved in 5 disulfide linkages. Endoprotease Lys-C digestion of purified PHMcc under nonreducing conditions cleaved the protein at Lys219, indicating that the protein consists of separable N- and C-terminal domains with internal disulfide linkages, that are connected by an exposed linker region. Disulfide-linked peptides generated by sequential CNBr and pepsin treatment of radiolabeled PHMcc were separated by reverse phase HPLC and identified by Edman degradation. Three disulfide linkages occur in the N-terminal domain (Cys47-Cys186, Cys81-Cys126, and Cys114-Cys131), along with three of the His residues critical to catalytic activity (His107, His108, and His172). Two disulfide linkages (Cys227-Cys334 and Cys293-Cys315) occur in the C-terminal domain, along with the remaining two essential His residues (His242, His244) and Met314, thought to be essential in binding one of the two nonequivalent copper atoms. Substitution of Tyr79 or Tyr318 with Phe increased the Km of PHM for its peptidylglycine substrate without affecting the Vmax. Replacement of Glu313 with Asp increased the Km 8-fold and decreased the kcat 7-fold, again identifying this region of the C-terminal domain as critical to catalytic activity. Taking into account information on the copper ligands in PHM, we propose a two-domain model with a copper site in each domain that allows spatial proximity between previously described copper ligands and residues identified as catalytically important.

Animals↗

PubMed1997

Kalirin, a cytosolic protein with spectrin-like and GDP/GTP exchange factor-like domains that interacts with peptidylglycine alpha-amidating monooxygenase, an integral membrane peptide-processing enzyme.

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 alpha-amidating monooxygenase (PAM). P-CIP10 is a 217-kDa cytosolic protein with nine spectrin-like 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.

Amino Acid Sequence↗