Kalirin Expression Research Articles

GDNF facilitates cognitive function recovery following neonatal surgical-induced learning and memory impairment via activation of the RET pathway and modulation of downstream effectors PKMζ and Kalirin in rats

ObjectiveThe aim of this study is to elucidate the underlying mechanism through which glial cell line-derived neurotrophic factor (GDNF) improves cognitive deficits in adults resulting from neonatal surgical interventions. MethodsNewborn Sprague–Dawley rats, regardless of gender, were randomly allocated into seven groups on postnatal day 7 as follows (n=15): (1) Control group (not subjected to anesthesia, surgery, or any pharmaceutical interventions); (2) GDNF group (received intracerebroventricular injection of GDNF); (3) Surgery group (underwent right carotid artery exposure under anesthesia with 3 % sevoflurane); (4) Surgery plus GDNF group; (5) Surgery plus GDNF and type II JAK inhibitor NVP-BBT594 (BBT594) group (administered intraperitoneal injection of BBT594); (6) BBT group; and (7) Surgery plus BBT group. Starting from postnatal day 33, all rats underwent Barnes maze and fear conditioning tests, followed by decapitation under sevoflurane anesthesia for subsequent analyses. The left hemibrains underwent Golgi staining, while the right hemibrains were used for hippocampal protein extraction to assess Protein kinase Mζ (PKMζ) and Kalirin expression through western blotting. ResultsGDNF demonstrated a mitigating effect on spatial learning and memory impairment, as well as context-related fear memory impairment, reductions in dendritic total lengths, and spinal density within the hippocampus induced by surgical intervention. Notably, all of these ameliorative effects of GDNF were reversed upon administration of the RET inhibitor BBT594. Additionally, GDNF alleviated the downregulation of protein expression of PKMζ and Kalirin in the hippocampus of rats subjected to surgery, subsequently reversed by BBT594. ConclusionThe effective impact of GDNF on learning and memory impairment caused by surgical intervention appears to be mediated through the RET pathway. Moreover, GDNF may exert its influence by upregulating the expression of PKMζ and Kalirin, consequently enhancing the development of dendrites and dendritic spines.

Kalirin is involved in epileptogenesis by modulating the activity of the Rac1 signaling pathway

Background and objectiveEpilepsy is a common chronic brain disease. Despite the availability of various anti-seizure drugs, approximately 30 % of patients do not respond to treatment. Recent research suggests that Kalirin plays a role in regulating neurological function. However, the pathogenesis of Kalirin in epileptic seizures remains unclear. This study aims to investigate the role and mechanism of Kalirin in epileptogenesis. Materials and methodsAn epileptic model was induced by intraperitoneal injection of pentylenetetrazole (PTZ). Endogenous Kalirin was inhibited using shRNA. The expression of Kalirin, Rac1, and Cdc42 in the hippocampal CA1 region was measured using Western blotting. Spine and synaptic structures were examined using Golgi staining and electron microscopy. Moreover, the necrotic neurons in CA1 were examined using HE staining. ResultsThe results indicated that the epileptic score increased in epileptic animals, while inhibition of Kalirin decreased the epileptic scores and increased the latent period of the first seizure attack. Inhibition of Kalirin attenuated the increases in Rac1 expression, dendritic spine density, and synaptic vesicle number in the CA1 region induced by PTZ. However, the increase in Cdc42 expression was not affected by the inhibition of Kalirin. ConclusionThis study suggests that Kalirin is involved in the development of seizures by modulating the activity of Rac1, providing a novel anti-epileptic target.

Role of Kalirin and mouse strain in retention of spatial memory training in an Alzheimer’s disease model mouse line

Nontransgenic and 3xTG transgenic mice, which express mutant transgenes encoding human amyloid precursor protein (hAPP) along with Alzheimer’s disease–associated versions of hTau and a presenilin mutation, acquired the Barnes Maze escape task equivalently at 3–9 months of age. Although nontransgenics retested at 6 and 9 months acquired the escape task more quickly than naïve mice, 3xTG mice did not. Deficits in Kalirin, a multidomain protein scaffold and guanine nucleotide exchange factor that regulates dendritic spines, has been proposed as a contributor to the cognitive decline observed in Alzheimer’s disease. To test whether deficits in Kalirin might amplify deficits in 3xTG mice, mice heterozygous/hemizygous for Kalirin and the 3xTG transgenes were generated. Mouse strain, age and sex affected cortical expression of key proteins. hAPP levels in 3xTG mice increased total APP levels at all ages. Kalirin expression showed strong sex-dependent expression in C57 but not B6129 mice. Decreasing Kalirin levels to half had no effect on Barnes Maze task acquisition or retraining in 3xTG hemizygous mice.

Kalirin Interacts with TRAPP and Regulates Rab11 and Endosomal Recycling.

Coordinated actions of Rab and Rho are necessary for numerous essential cellular processes ranging from vesicle budding to whole cell movement. How Rab and Rho are choreographed is poorly understood. Here, we report a protein complex comprised of kalirin, a Rho guanine nucleotide exchange factor (GEF) activating Rac1, and RabGEF transport protein particle (TRAPP). Kalirin was identified in a mass spectrometry analysis of proteins precipitated by trappc4 and detected on membranous organelles containing trappc4. Acute knockdown of kalirin did not affect trappc4, but significantly reduced overall and membrane-bound levels of trappc9, which specifies TRAPP toward activating Rab11. Trappc9 deficiency led to elevated expression of kalirin in neurons. Co-localization of kalirin and Rab11 occurred at a low frequency in NRK cells under steady state and was enhanced upon expressing an inactive Rab11 mutant to prohibit the dissociation of Rab11 from the kalirin-TRAPP complex. The small RNA-mediated depletion of kalirin diminished activities in cellular membranes for activating Rab11 and resulted in a shift in size of Rab11 positive structures from small to larger ones and tubulation of recycling endosomes. Our study suggests that kalirin and TRAPP form a dual GEF complex to choreograph actions of Rab11 and Rac1 at recycling endosomes.

Using Kalirin conditional knockout mice to distinguish its role in dopamine receptor mediated behaviors

BackgroundMice lacking Kalirin-7 (Kal7KO), a Rho GDP/GTP exchange factor, self-administer cocaine at a higher rate than wildtype mice, and show an exaggerated locomotor response to experimenter-administered cocaine. Kal7, which localizes to post-synaptic densities at glutamatergic synapses, interacts directly with the GluN2B subunit of the N-methyl-d-aspartate (NMDA; GluN) receptor. Consistent with these observations, Kal7 plays an essential role in NMDA receptor dependent long term potentiation and depression, and glutamatergic transmission plays a key role in the response to chronic cocaine. A number of genetic studies have implicated altered Kalirin expression in schizophrenia and other disorders such as Alzheimer’s Disease.ResultsA comparison of the effects of experimenter-administered cocaine on mice lacking all Kalirin isoforms to its effects on mice lacking only Kalirin-7 identified Kal7 as the key isoform whose deletion produces exaggerated locomotor responses to cocaine. Pretreatment of Kal7KO mice with a low dose of ifenprodil, a selective GluN2B antagonist, eliminated their enhanced locomotor response to cocaine, revealing an important role for GluN2B in this behavior. Selective knockout of Kalirin in dopamine transporter expressing neurons produced a transient enhancement of cocaine-induced locomotion, while knockout of Kalirin in Drd1a- or Drd2-dopamine receptor expressing neurons was without effect. As observed in Kalirin global knockout mice, eliminating Kalirin expression in Drd2-expressing neurons increased exploratory behavior in the elevated zero maze, an effect eliminated by pretreatment with ifenprodil.ConclusionsThe cocaine-sensitive neuronal pathways which are most sensitive to altered Kalirin function may be the pathways most dependent on GluN2B and Drd2.

A Novel Long Non-coding RNA, durga Modulates Dendrite Density and Expression of kalirin in Zebrafish.

Kalirin, a key player in axonal development, nerve growth and synaptic re-modeling, is implicated in many pathological conditions like schizophrenia and autism-spectrum disorders. Alternative promoters and splicing lead to functionally distinct isoforms, but the post-transcriptional regulation of Kalirin has not been studied. Here, we report a novel non-coding RNA, which we name durga, arising from the first exon of kalirin a (kalrna) in the antisense orientation in zebrafish. The kalrna and durga transcripts are barely detectable during early development, but steadily increase by 24 hours post-fertilization (hpf) as the brain develops. Over-expression of durga in the zebrafish embryo led to an increase in kalrna expression. The morphology of the neurons cultured from durga injected embryos had significantly fewer and shorter dendrites. Although durga has no apparent sequence homolog in mammals, based on gene synteny, we found a non-coding RNA arising from the 5′ end of the human Kalrn gene and expressed in the human neuronal cell line, SH-SY5Y. We propose that the zebrafish lncRNA durga maintains dendritic length and density through regulation of kalrna expression and this may have further implications in mammalian systems.

A sequence variant in human KALRN impairs protein function and coincides with reduced cortical thickness.

Dendritic spine pathology is a key feature of several neuropsychiatric disorders. The Rac1 guanine nucleotide exchange factor kalirin-7 is critical for spine morphogenesis on cortical pyramidal neurons. Here we identify a rare coding variant in the KALRN gene region that encodes the catalytic domain, in a schizophrenia patient and his sibling with major depressive disorder. The D1338N substitution significantly diminished the protein's ability catalyze the activation of Rac1. Contrary to wild-type kalirin-7, kalirin-7-D1338N failed to increase spine size and density. Both subjects carrying the polymorphism displayed reduced cortical volume in the superior temporal sulcus (STS), a region implicated in schizophrenia. Consistent with this, mice with reduced kalirin expression showed reduced neuropil volume in the rodent homolog of the STS. These data suggest that single amino acid changes in proteins involved in dendritic spine function can have significant effects on the structure and function of the cerebral cortex.

The Rho-GEF Kalirin regulates bone mass and the function of osteoblasts and osteoclasts

Bone homeostasis is maintained by the balance between bone resorption by osteoclasts and bone formation by osteoblasts. Dysregulation in the activity of the bone cells can lead to osteoporosis, a disease characterized by low bone mass and an increase in bone fragility and risk of fracture. Kalirin is a novel GTP-exchange factor protein that has been shown to play a role in cytoskeletal remodeling and dendritic spine formation in neurons. We examined Kalirin expression in skeletal tissue and found that it was expressed in osteoclasts and osteoblasts. Furthermore, micro-CT analyses of the distal femur of global Kalirin knockout (Kal-KO) mice revealed significantly reduced trabecular and cortical bone parameters in Kal-KO mice, compared to WT mice, with significantly reduced bone mass in 8, 14 and 36week-old female Kal-KO mice. Male mice also exhibited a decrease in bone parameters but not to the level seen in female mice. Histomorphometric analyses also revealed decreased bone formation rate in 14week-old female Kal-KO mice, as well as decreased osteoblast number/bone surface and increased osteoclast surface/bone surface. Consistent with our in vivo findings, the bone resorbing activity and differentiation of Kal-KO osteoclasts was increased in vitro. Although alkaline phosphatase activity by Kal-KO osteoblasts was increased in vitro, Kal-KO osteoblasts showed decreased mineralizing activity, as well as decreased secretion of OPG, which was inversely correlated with ERK activity. Taken together, our findings suggest that deletion of Kalirin directly affects osteoclast and osteoblast activity, leading to decreased OPG secretion by osteoblasts which is likely to alter the RANKL/OPG ratio and promote osteoclastogenesis. Therefore, Kalirin may play a role in paracrine and/or endocrine signaling events that control skeletal bone remodeling and the maintenance of bone mass.

RACing up a New Regulatory Mechanism for Vascular Smooth Muscle Cell Migration

Pathophysiological vascular smooth muscle cell (VSMC) migration is a critical component of atherosclerosis and contributes substantially to neointimal hyperplasia. Injury to the intimal layer of the vessel promotes the dedifferentiation of VSMCs in the tunica media from a quiescent contractile phenotype to a synthetic phenotype that proliferates and migrates, thus contributing to neointimal thickening. A better understanding of the signaling mechanisms that promote VSMC dedifferentiation, proliferation, and migration may lead to the identification of new pharmacological targets for atherosclerosis and other vascular diseases. See accompanying article on page 702 Several signal transduction pathways regulate actin polymerization and cell contractility, both integral to VSMC migration. The process of cell migration is complex but requires fundamental processes that depend on tight regulation of the GTPases Rac and Rho (Figure).1–3 Migration initiates when a cell is exposed to a chemoattractant gradient and establishes polarity. This triggers the extension of the plasma membrane, called a lamellipodium, in the direction of eventual cell movement and establishes the front/leading edge of the cell.2,4 In vascular injury, a gradient is often established by the release of platelet-derived growth factor (PDGF), such that VSMCs migrate toward the lumen of the vessel.4,5 The formation of lamellipodia requires the polymerization and assembly of actin, which is regulated by the GTPase Rac. …

Tau phosphorylation is exaggerated in Alzheimer disease with psychosis

The occurrence of psychotic symptoms in Alzheimer's disease subjects (AD+P) represents a subgroup characterized by more severe impairment and more rapid decline in cognition compared to AD without psychosis (AD-P). AD+P aggregates within families with an estimated heritability of 61%, indicating a substantial genetic component. Since the strongest correlate of cognitive impairment in AD is synapse loss across cortical regions, we hypothesize that the genetic architecture of AD+P leads to exaggerated synaptic vulnerability. In dorsolateral prefrontal cortex (DLPFC) we previously found increased disruption of synapse integrity, lower kalirin expression, and greater Aβ42/Aβ40 ratio in AD+P cases. Since hyperphosphorylated tau may be a necessary mediator of Aβ-induced synaptic toxicity, we evaluated DLPFC grey matter phospho-tau (pTau) in a well-matched cohort of 26 AD+P and 19 AD-P cases. We used AT8, an antibody for a triple phosphorylation site epitope (ser199/ser202/thr205) on pTau-associated dystrophic neurites, neuropil threads, and neurofibrillary tangles, and assessed both mean fluorescence intensity, indicative of relative pTau concentration (pTC), and pTau volume fraction (pTVV; fraction of total grey matter volume occupied by pTau) in the DLPFC. Both of our measures were significantly correlated with Braak stage (pTC rs = 0.491, p < 0.001; pTVV rs = 0.758, p < 0.001). In AD+P, we found that pTC was greater (p = 0.006) and that pTVV trended toward an increase (p = 0.082). We then evaluated whether pTC and/or pTVV affected cognitive impairment by examining the most recent scores on the mini-mental status exam (MMSE), as an indication of global cognitive function, and on the digit span backwards test (DSB), as an indicator of cognitive function associated specifically with the DLPFC. Consistent with previous findings, AD+P cases scored lower on the mini-mental status exam (MMSE; p = 0.026), despite similar durations of illness in the two groups. Lower scores on the MMSE were influenced by higher pTC (p = 0.006), and pTVV (p = 0.030); lower scores on the DSB were also influenced by pTC (p = 0.013) and pTVV (p = 0.008). These novel findings indicate that while higher pTC and pTVV are associated with worse cognitive impairment, pTC is specifically higher in AD+P. This suggests that phosphorylation of tau may be exaggerated in AD+P, which along with our previous findings, points to more severe pathological dynamics in AD+P.

β-Amyloid 42/40 ratio and kalirin expression in Alzheimer disease with psychosis

Psychosis in Alzheimer disease differentiates a subgroup with more rapid decline, is heritable, and aggregates within families, suggesting a distinct neurobiology. Evidence indicates that greater impairments of cerebral cortical synapses, particularly in dorsolateral prefrontal cortex, may contribute to the pathogenesis of psychosis in Alzheimer disease (AD) phenotype. Soluble β-amyloid induces loss of dendritic spine synapses through impairment of long-term potentiation. In contrast, the Rho guanine nucleotide exchange factor (GEF) kalirin is an essential mediator of spine maintenance and growth in cerebral cortex. We therefore hypothesized that psychosis in AD would be associated with increased soluble β-amyloid and reduced expression of kalirin in the cortex. We tested this hypothesis in postmortem cortical gray matter extracts from 52 AD subjects with and without psychosis. In subjects with psychosis, the β-amyloid1–42/β-amyloid1–40 ratio was increased, due primarily to reduced soluble β-amyloid1–40, and kalirin-7, -9, and -12 were reduced. These findings suggest that increased cortical β-amyloid1–42/β-amyloid1–40 ratio and decreased kalirin expression may both contribute to the pathogenesis of psychosis in AD.

Kalrn promoter usage and isoform expression respond to chronic cocaine exposure

BackgroundThe long-term effects of cocaine on behavior are accompanied by structural changes in excitatory glutamatergic synapses onto the medium spiny neurons of the striatum. The Kalrn gene encodes several functionally distinct isoforms; these multidomain guanine nucleotide exchange factors (GEFs) contain additional domains known to interact with phosphatidylinositides as well as with a number of different proteins. Through their activation of Rho proteins and their interactions with other proteins, the different Kalirin isoforms affect cytoskeletal organization. Chronic exposure of adult male rodents to cocaine increases levels of Kalirin 7 in the striatum. When exposed chronically to cocaine, mice lacking Kalirin 7, the major adult isoform, fail to show an increase in dendritic spine density in the nucleus accumbens, show diminished place preference for cocaine, and exhibit increased locomotor activity in response to cocaine.ResultsThe use of alternate promoters and 3'-terminal exons of the mouse Kalrn gene were investigated using real-time quantitative polymerase chain reaction. While the two most distal full-length Kalrn promoters are used equally in the prefrontal cortex, the more proximal of these promoters accounts for most of the transcripts expressed in the nucleus accumbens. The 3'-terminal exon unique to the Kalirin 7 isoform accounts for a greater percentage of the Kalrn transcripts in prefrontal cortex than in nucleus accumbens. Western blot analyses confirmed these differences. Chronic cocaine treatment increases usage of the promoter encoding the Δ-Kalirin isoforms but does not alter full-length Kalirin promoter usage. Usage of the 3'-terminal exon unique to Kalirin 7 increases following chronic cocaine exposure.ConclusionsKalrn promoter and 3'-terminal exon utilization are region-specific. In the nucleus accumbens, cocaine-mediated alterations in promoter usage and 3'-terminal exon usage favor expression of Kalirin 7 and Δ-Kalirin 7. The Δ-isoform, which lacks a Sec14p domain and four of the nine spectrin-like repeats found in full-length Kalirin isoforms, increases spine headsize without increasing dendritic spine numbers. Thus cocaine-mediated changes in alternative splicing of the Kalrn gene may contribute importantly to the behavioral, morphological and biochemical responses observed.

Kalirin-7 Controls Activity-Dependent Structural and Functional Plasticity of Dendritic Spines

Activity-dependent rapid structural and functional modifications of central excitatory synapses contribute to synapse maturation, experience-dependent plasticity, and learning and memory and are associated with neurodevelopmental and psychiatric disorders. However, the signal transduction mechanisms that link glutamate receptor activation to intracellular effectors that accomplish structural and functional plasticity are not well understood. Here we report that NMDA receptor activation in pyramidal neurons causes CaMKII-dependent phosphorylation of the guanine-nucleotide exchange factor (GEF) kalirin-7 at residue threonine 95, regulating its GEF activity, leading to activation of small GTPase Rac1 and rapid enlargement of existing spines. Kalirin-7 also interacts with AMPA receptors and controls their synaptic expression. By demonstrating that kalirin expression and spine localization are required for activity-dependent spine enlargement and enhancement of AMPAR-mediated synaptic transmission, our study identifies a signaling pathway that controls structural and functional spine plasticity.

Critical Role for Kalirin in Nerve Growth Factor Signaling through TrkA

Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kalirin expression using antisense RNA decreased nerve growth factor (NGF)-induced TrkA autophosphorylation and process extension. Kalirin overexpression potentiated neurotrophin-stimulated TrkA autophosphorylation and neurite outgrowth in PC12 cells at a low concentration of NGF. Furthermore, elevated Kalirin expression resulted in catalytic activation of TrkA, as demonstrated by in vitro kinase assays and increased NGF-stimulated cellular activation of Rac, Mek, and CREB. Domain mapping demonstrated that the N-terminal Kalirin pleckstrin homology domain mediates the interaction with TrkA. The effects of Kalirin on TrkA provide a molecular basis for the requirement of Kalirin in process extension from PC12 cells and for previously observed effects on axonal extension and dendritic maintenance. The interaction of TrkA with the pleckstrin homology domain of Kalirin may be one example of a general mechanism whereby receptor/Rho GEF pairings play an important role in receptor tyrosine kinase activation and signal transduction.

Kalirin Expression Is Regulated by Multiple Promoters

Kalirin is a multidomain guanine nucleotide exchange factor for small GTPbinding proteins of the Rho family. It is expressed in multiple isoforms that contain different combinations of functional domains and display a complex pattern of expression during brain development. In addition to the isoforms generated through alternative splicing, we have identified multiple transcriptional start sites in rats and humans. These multiple transcriptional start sites result in full-length Kalirin transcripts possessing different 5' ends encoding proteins with differing amino termini. These alternative first exons display different patterns of expression in developing rats and humans and in cultured cells. Most of these alternate first exons lie >100 kb upstream of exon 2 in both rats and humans. Comparisons of the rat and human Kalirin promoter regions reveal numerous shared potential regulatory elements.

Kalirin, a Multifunctional Rho Guanine Nucleotide Exchange Factor, Is Necessary for Maintenance of Hippocampal Pyramidal Neuron Dendrites and Dendritic Spines

The structures of dendritic spines and the dendritic tree, key determinants of neuronal function, are regulated by diverse inputs that affect many scaffolding and signaling molecules. Nevertheless, here we show that reduced expression of a single gene results in loss of dendritic spines and a decrease in dendritic complexity. Kalirin, a dual Rho GDP-GTP exchange factor, causes spine formation when overexpressed. Reduced expression of Kalirin in CA1 hippocampal neurons resulted in a reduction in linear spine density, with dispersion of postsynaptic density markers and elimination of presynaptic endings. Simplification of the apical dendritic tree preceded simplification of basal dendrites. Pyramidal cell axons were not dramatically altered. Although many factors determine dendrite shape and spine formation, expression of Kalirin is necessary for the normal function of these many regulatory elements.

Kalirin, a GDP/GTP exchange factor of the Dbl family, is localized to nerve, muscle, and endocrine tissue during embryonic rat development.

Kalirin, a homologue of trio and UNC-73, has been previously demonstrated to cause cytoskeletal rearrangements, enhanced outgrowth of neuritic processes, and altered secretion. In the adult rat, kalirin is specifically localized to the central nervous system, with the main adult isoform, kalirin-7, concentrated in neuronal postsynaptic densities. In this study we examined the expression of kalirin in rat tissue from embryonic Day 10 (E10) through E18, using an antibody that detects all known kalirin isoforms. Kalirin expression in the embryo was more widespread than in the adult, with localization of kalirin protein to both neuronal and non-neuronal tissue, such as muscle, lung, intestinal epithelium, and pancreas. In neurons, kalirin was localized both in cell bodies and axon processes; in muscle tissue, kalirin was highly localized to migrating myogenic cells and at muscle attachment sites. Western blotting analysis indicated that kalirin-7, the major adult isoform, was a minor component of embryonic kalirin; the main isoform expressed in the embryo was kalirin-9. This is the first identification of kalirin expression in embryonic tissue and the first demonstration of non-neuronal expression of kalirin. (J Histochem Cytochem 49:833-844, 2001)

Expression of Kalirin, a neuronal GDP/GTP exchange factor of the Trio family, in the central nervous system of the adult rat

Kalirin is a multifunctional protein identified by its interaction with peptidylglycine alpha-amidating monooxygenase, an enzyme essential for neuropeptide biosynthesis. Several forms of Kalirin exist, all containing spectrin-like repeats, a Dbl homology (DH) domain, and an adjacent pleckstrin homology (PH) domain; several different COOH-termini provide additional DH/PH domains and a putative protein kinase. Kalirin binds Rac1 and affects cytoskeletal organization, neuropeptide secretion, and iNOS activity. By in situ hybridization, the highest levels of Kalirin mRNA were found in the cerebral cortex, hippocampal formation, and Purkinje cells, with high levels also in thalamus, caudate putamen, septal nucleus, nucleus accumbens, amygdala, and anterior olfactory nucleus. Low levels of Kalirin mRNA were detected in the paraventricular, supraoptic, and reticular thalamic nuclei and in the ventromedial hypothalamic nucleus. Brain areas with high levels of Kalirin mRNA showed strong Kalirin-like immunoreactivity. Pyramidal neurons with strongly staining soma and long dendrites were observed primarily in layer 5 of the cerebral cortex. In the hippocampus, a uniform distribution of neurons with fine dendritic staining was observed in the pyramidal cell layer, in the granule cell layer, and in the hilar cells of the dentate gyrus as well as in isolated interneurons. Cerebellar Purkinje neurons exhibited intense staining in the soma and in extensive dendritic arbors extending to the surface of the molecular layer. During embryonic development, Trio, the Drosophila orthologue of Kalirin, plays an essential role in axon guidance; localization of Kalirin to the somatodendritic region of adult neurons provides the basis for future studies of regulation and function.

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.