Germline loss-of-function PAM variants are enriched in subjects with pituitary hypersecretion.

Introduction: Pituitary adenomas (PAs) are common, usually benign tumors of the anterior pituitary gland which, for the most part, have no known genetic cause. PAs are associated with major clinical effects due to hormonal dysregulation and tumoral impingement on vital brain structures. PAM encodes a multifunctional protein responsible for the essential C-terminal amidation of secreted peptides. Methods: Following the identification of a loss-of-function variant (p.Arg703Gln) in the peptidylglycine a-amidating monooxygenase (PAM) gene in a family with pituitary gigantism, we investigated 299 individuals with sporadic PAs and 17 familial isolated PA kindreds for PAM variants. Genetic screening was performed by germline and tumor sequencing and germline copy number variation (CNV) analysis. Results: In germline DNA, we detected seven heterozygous, likely pathogenic missense, truncating, and regulatory SNVs. These SNVs were found in sporadic subjects with growth hormone excess (p.Gly552Arg and p.Phe759Ser), pediatric Cushing disease (c.-133T>C and p.His778fs), or different types of PAs (c.-361G>A, p.Ser539Trp, and p.Asp563Gly). The SNVs were functionally tested in vitro for protein expression and trafficking by Western blotting, splicing by minigene assays, and amidation activity in cell lysates and serum samples. These analyses confirmed a deleterious effect on protein expression and/or function. By interrogating 200,000 exomes from the UK Biobank, we confirmed a significant association of the PAM gene and rare PAM SNVs with diagnoses linked to pituitary gland hyperfunction. Conclusion: The identification of PAM as a candidate gene associated with pituitary hypersecretion opens the possibility of developing novel therapeutics based on altering PAM function.

Germline loss-of-function PAM variants are enriched in subjects with pituitary hypersecretion.

Pituitary adenomas (PAs) are common, usually benign tumors of the anterior pituitary gland which, for the most part, have no known genetic cause. PAs are associated with major clinical effects due to hormonal dysregulation and tumoral impingement on vital brain structures. Following the identification of a loss-of-function variant (p.Arg703Gln) in the PAM gene in a family with pituitary gigantism, we investigated 299 individuals with sporadic PAs and 17 familial isolated pituitary adenomas kindreds for PAM variants. PAM encodes a multifunctional protein responsible for the essential C-terminal amidation of secreted peptides. Genetic screening was performed by germline and tumor sequencing and germline copy number variation (CNV) analysis. No germline CNVs or somatic single nucleotide variants (SNVs) were identified. We detected seven likely pathogenic heterozygous missense, truncating, and regulatory SNVs. These SNVs were found in sporadic subjects with GH excess (p.Gly552Arg and p.Phe759Ser), pediatric Cushing disease (c.-133T>C and p.His778fs), or with different types of PAs (c.-361G>A, p.Ser539Trp, and p.Asp563Gly). The SNVs were functionally tested in vitro for protein expression and trafficking by Western blotting, for splicing by minigene assays, and for amidation activity in cell lysates and serum samples. These analyses confirmed a deleterious effect on protein expression and/or function. By interrogating 200,000 exomes from the UK Biobank, we confirmed a significant association of the PAM gene and rare PAM SNVs to diagnoses linked to pituitary gland hyperfunction. Identification of PAM as a candidate gene associated with pituitary hypersecretion opens the possibility of developing novel therapeutics based on altering PAM function.

Structure of the Sec14 domain of Kalirin reveals a distinct class of lipid-binding module in RhoGEFs.

Gated entry of lipophilic ligands into the enclosed hydrophobic pocket in stand-alone Sec14 domain proteins often links lipid metabolism to membrane trafficking. Similar domains occur in multidomain mammalian proteins that activate small GTPases and regulate actin dynamics. The neuronal RhoGEF Kalirin, a central regulator of cytoskeletal dynamics, contains a Sec14 domain (KalbSec14) followed by multiple spectrin-like repeats and catalytic domains. Previous studies demonstrated that Kalirin lacking its Sec14 domain fails to maintain cell morphology or dendritic spine length, yet whether and how KalbSec14 interacts with lipids remain unknown. Here, we report the structural and biochemical characterization of KalbSec14. KalbSec14 adopts a closed conformation, sealing off the canonical ligand entry site, and instead employs a surface groove to bind a limited set of lysophospholipids. The low-affinity interactions of KalbSec14 with lysolipids are expected to serve as a general model for the regulation of Rho signaling by other Sec14-containing Rho activators.

Regulated processing and secretion of a peptide precursor in cilia.

Cilia are sensory and secretory organelles that both receive information from the environment and transmit signals. Cilia-derived vesicles (ectosomes), formed by outward budding of the ciliary membrane, carry enzymes and other bioactive products; this process represents an ancient mode of regulated secretion. Peptidergic intercellular communication controls a wide range of physiological and behavioral responses and occurs throughout eukaryotes. The Chlamydomonas reinhardtii genome encodes what appear to be numerous prepropeptides and enzymes homologous to those used to convert metazoan prepropeptides into bioactive peptide products. Since C. reinhardtii, a green alga, lack the dense core vesicles in which metazoan peptides are processed and stored, we explored the hypothesis that propeptide processing and secretion occur through the regulated release of ciliary ectosomes. A synthetic peptide (GATI-amide) that could be generated from a 91-kDa peptide precursor (proGATI) serves as a chemotactic modulator, attracting minus gametes while repelling plus gametes. Here we dissect the processing pathway that leads to formation of an amidated peptidergic sexual signal specifically on the ciliary ectosomes of plus gametes. Unlike metazoan propeptides, modeling studies identified stable domains in proGATI. Mass spectrometric analysis of a potential prohormone convertase and the amidated proGATI-derived products found in cilia and mating ectosomes link endoproteolytic cleavage to ectosome entry. Extensive posttranslational modification of proGATI confers stability to its amidated product. Analysis of this pathway affords insight into the evolution of peptidergic signaling; this will facilitate studies of the secretory functions of metazoan cilia.

Peptidylglycine α-amidating monooxygenase as a therapeutic target or biomarker for human diseases.

Peptides play a key role in controlling many physiological and neurobiological pathways. Many bioactive peptides require a C-terminal α-amide for full activity. The bifunctional enzyme catalysing α-amidation, peptidylglycine α-amidating monooxygenase (PAM), is the sole enzyme responsible for amidated peptide biosynthesis, from Chlamydomonas reinhardtii to Homo sapiens. Many neuronal and endocrine functions are dependent upon amidated peptides; additional amidated peptides are growth promoters in tumours. The amidation reaction occurs in two steps, glycine α-hydroxylation followed by dealkylation to generate the α-amide product. Currently, most potentially useful inhibitors target the first reaction, which is rate-limiting. PAM is a membrane-bound enzyme that visits the cell surface during peptide secretion. PAM is then used again in the biosynthetic pathway, meaning that cell-impermeable inhibitors or inactivators could have therapeutic value for the treatment of cancer or psychiatric abnormalities. To date, inhibitor design has not fully exploited the structures and mechanistic details of PAM.

PAM: diverse roles in neuroendocrine cells, cardiomyocytes, and green algae.

Our understanding of the ways in which peptides are used for communication in the nervous and endocrine systems began with the identification of oxytocin, vasopressin, and insulin, each of which is stored in electron-dense granules, ready for release in response to an appropriate stimulus. For each of these peptides, entry of its newly synthesized precursor into the ER lumen is followed by transport through the secretory pathway, exposing the precursor to a sequence of environments and enzymes that produce the bioactive products stored in mature granules. A final step in the biosynthesis of many peptides is C-terminal amidation by peptidylglycine α-amidating monooxygenase (PAM), an ascorbate- and copper-dependent membrane enzyme that enters secretory granules along with its soluble substrates. Biochemical and cell biological studies elucidated the highly conserved mechanism for amidated peptide production and raised many questions about PAM trafficking and the effects of PAM on cytoskeletal organization and gene expression. Phylogenetic studies and the discovery of active PAM in the ciliary membranes of Chlamydomonas reinhardtii, a green alga lacking secretory granules, suggested that a PAM-like enzyme was present in the last eukaryotic common ancestor. While the catalytic features of human and C. reinhardtii PAM are strikingly similar, the trafficking of PAM in C. reinhardtii and neuroendocrine cells and secretion of its amidated products differ. A comparison of PAM function in neuroendocrine cells, atrial myocytes, and C. reinhardtii reveals multiple ways in which altered trafficking allows PAM to accomplish different tasks in different species and cell types.

Multiple roles for peptidylglycine α-amidating monooxygenase in the response to hypoxia.

The biosynthesis of many of the peptides involved in homeostatic control requires peptidylglycine α-amidating monooxygenase (PAM), an ancient, highly conserved copper- and ascorbate-dependent enzyme. Using the production of amidated chromogranin A to monitor PAM function in tumor cells, physiologically relevant levels of hypoxia were shown to inhibit this monooxygenase. The ability of primary pituitary cells exposed to hypoxic conditions for 4 h to produce amidated chromogranin A was similarly inhibited. The affinity of the purified monooxygenase for oxygen (Km = 99 ± 19 µM) was consistent with this result. The ability of PAM to alter secretory pathway behavior under normoxic conditions required its monooxygenase activity. Under normoxic conditions, hypoxia-inducible factor 1a levels in dense cultures of corticotrope tumor cells expressing high levels of PAM exceeded those in control cells; expression of inactive monooxygenase did not have this effect. The effects of hypoxia on levels of two PAM-regulated genes (activating transcription factor 3 [Atf3] and FK506 binding protein 2 [Fkbp2]) differed in cells expressing high versus low levels of PAM. Putative hypoxia response elements occur in both human and mouse PAM, and hPAM has consistently been identified as one of the genes upregulated in response to hypoxia. Expression of PAM is also known to alter gene expression. A quarter of the genes consistently upregulated in response to hypoxia were downregulated following increased expression of PAM. Taken together, our data suggest roles for PAM and amidated peptide secretion in the coordination of tissue-specific responses to hypoxia.

Cell-type specific knockout of peptidylglycine α-amidating monooxygenase reveals specific behavioral roles in excitatory forebrain neurons and cardiomyocytes.

Neuropeptides and peptide hormones play a crucial role in integrating the many factors that affect physiologic and cognitive processes. The potency of many of these peptides requires an amidated amino acid at the C-terminus; a single enzyme, peptidylglycine α-amidating monooxygenase (PAM), catalyzes this modification. Anxiety-like behavior is known to be altered in mice with a single functional Pam allele (Pam+/- ) and in mice unable to express Pam in excitatory forebrain neurons (PamEmx1-cKO/cKO ) or in cardiomyocytes (PamMyh6-cKO/cKO ). Examination of PAM-positive and glutamic acid decarboxylase 67 (GAD)-positive cells in the amygdala of PamEmx1-cKO/cKO mice demonstrated the absence of PAM in pyramidal neurons and its continued presence in GAD-positive interneurons, suggestive of altered excitatory/inhibitory balance. Additional behavioral tests were used to search for functional alterations in these cell-type specific knockout mice. PamEmx1-cKO/cKO mice exhibited a less focused search pattern for the Barnes Maze escape hole than control or PamMyh6-cKO/cKO mice. While wildtype mice favor interacting with novel objects as opposed to familiar objects, both PamEmx1-cKO/cKO and PamMyh6-cKO/cKO mice exhibited significantly less interest in the novel object. Since PAM levels in the central nervous system of PamMyh6-cKO/cKO mice are unaltered, the behavioral effect observed in these mice may reflect their inability to produce atrial granules and the resulting reduction in serum levels of atrial natriuretic peptide. In the sociability test, male mice of all three genotypes spent more time with same-sex stranger mice; while control females showed no preference for stranger mice, female PamEmx1-cKO/cKO mice showed preference for same-sex stranger mice in all trials.

Commentary on: Peptidylglycine α-amidating Monooxygenase is Required for Atrial Secretory Granule Formation.

The electron-dense spherical granules found in the perinuclear region of atrial myocytes store and release both proatrial and probrain natriuretic peptides (proANP and proBNP, respectively). Mature ANP and BNP produce vasodilation and natriuresis and inhibit the renin-angiotensin and sympathetic nervous systems. Although neither ANP nor BNP is a-amidated, Peptidylglycine a-Amidating Monooxygenase (PAM), an integral membrane enzyme known to catalyze the a-amidation of peptidylglycine precursors, is the major atrial granule membrane protein. Selective deletion of PAM from cardiomyocytes impairs their ability to store proANP, resulting in an increase in proANP secretion. Exogenous expression of active or inactive PAM protein restores the ability of atrial myocytes to store proANP, leading to the suggestion that PAM functions as a cargo receptor for newly synthesized proANP.

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.

Peptidylglycine α-amidating monooxygenase is required for atrial secretory granule formation.

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is peptidylglycine α-amidating monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (PamMyh6-cKO/cKO) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult PamMyh6-cKO/cKO atria revealed a 13-fold drop in the number of secretory granules. When primary cultures of Pam0-Cre-cKO/cKO atrial myocytes (no Cre recombinase, PAM floxed) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in ANP precursor (proANP) levels. Expression of exogenous PAM in PamMyh6-cKO/cKO atrial myocytes produced a dose-dependent rescue of proANP content; strikingly, this response did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact. A threefold increase in the basal rate of proANP secretion by PamMyh6-cKO/cKO myocytes was a major contributor to its reduced levels. While proANP secretion was increased following treatment of control cultures with drugs that block the activation of Golgi-localized Arf proteins and COPI vesicle formation, proANP secretion by PamMyh6-cKO/cKO myocytes was unaffected. In cells lacking secretory granules, expression of exogenous PAM led to the accumulation of fluorescently tagged proANP in the cis-Golgi region. Our data indicate that COPI vesicle-mediated recycling of PAM from the cis-Golgi to the endoplasmic reticulum plays an essential role in the biogenesis of proANP containing atrial granules.

Correction to: PAM haploinsufficiency does not accelerate the development of diet and human IAPP-induced diabetes in mice.

Unfortunately, the human islet checklist was omitted from the electronic supplementary material (ESM) linked to this paper.

PAM haploinsufficiency does not accelerate the development of diet- and human IAPP-induced diabetes in mice.

AIMS/HYPOTHESIS: Peptide hormones are first synthesised as larger, inactive precursors that are converted to their active forms by endopeptidase cleavage and post-translational modifications, such as amidation. Recent, large-scale genome-wide studies have suggested that two coding variants of the amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), are associated with impaired insulin secretion and increased type 2 diabetes risk. We aimed to elucidate the role of PAM in modulating beta cell peptide amidation, beta cell function and the development of diabetes. METHODS: PAM transcript and protein levels were analysed in mouse islets following induction of endoplasmic reticulum (ER) or cytokine stress, and PAM expression patterns were examined in human islets. To study whether haploinsufficiency of PAM accelerates the development of diabetes, Pam+/- and Pam+/+ mice were fed a low-fat diet (LFD) or high-fat diet (HFD) and glucose homeostasis was assessed. Since aggregates of the PAM substrate human islet amyloid polypeptide (hIAPP) lead to islet inflammation and beta cell failure, we also investigated whether PAM haploinsufficiency accelerated hIAPP-induced diabetes and islet amyloid formation in Pam+/- and Pam+/+ mice with beta cell expression of hIAPP. RESULTS: Immunostaining revealed high expression of PAM in alpha, beta and delta cells in human pancreatic islets. Pam mRNA and PAM protein expression were reduced in mouse islets following administration of an HFD, and in isolated islets following induction of ER stress with thapsigargin, or cytokine stress with IL-1ß, IFN-γ and TFN-α. Despite Pam+/- only having 50% PAM expression and enzyme activity as compared with Pam+/+ mice, glucose tolerance and body mass composition were comparable in the two models. After 24 weeks of HFD, both Pam+/- and Pam+/+ mice had insulin resistance and impaired glucose tolerance, but no differences in glucose tolerance, insulin sensitivity or plasma insulin levels were observed in PAM haploinsufficient mice. Islet amyloid formation and beta cell function were also similar in Pam+/- and Pam+/+ mice with beta cell expression of hIAPP. CONCLUSIONS/INTERPRETATION: Haploinsufficiency of PAM in mice does not accelerate the development of diet-induced obesity or hIAPP transgene-induced diabetes.

Cilia-based peptidergic signaling.

Peptide-based intercellular communication is a ubiquitous and ancient process that predates evolution of the nervous system. Cilia are essential signaling centers that both receive information from the environment and secrete bioactive extracellular vesicles (ectosomes). However, the nature of these secreted signals and their biological functions remain poorly understood. Here, we report the developmentally regulated release of the peptide amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), and the presence of peptidergic signaling machinery (including propeptide precursors, subtilisin-like prohormone convertases, amidated products, and receptors) in ciliary ectosomes from the green alga Chlamydomonas. One identified amidated PAM product serves as a chemoattractant for mating-type minus gametes but repels plus gametes. Thus, cilia provide a previously unappreciated route for the secretion of amidated signaling peptides. Our study in Chlamydomonas and the presence of PAM in mammalian cilia suggest that ciliary ectosome-mediated peptidergic signaling dates to the early eukaryotes and plays key roles in metazoan physiology.

Identifying roles for peptidergic signaling in mice.

Despite accumulating evidence demonstrating the essential roles played by neuropeptides, it has proven challenging to use this information to develop therapeutic strategies. Peptidergic signaling can involve juxtacrine, paracrine, endocrine, and neuronal signaling, making it difficult to define physiologically important pathways. One of the final steps in the biosynthesis of many neuropeptides requires a single enzyme, peptidylglycine α-amidating monooxygenase (PAM), and lack of amidation renders most of these peptides biologically inert. PAM, an ancient integral membrane enzyme that traverses the biosynthetic and endocytic pathways, also affects cytoskeletal organization and gene expression. While mice, zebrafish, and flies lacking Pam (PamKO/KO ) are not viable, we reasoned that cell type-specific elimination of Pam expression would generate mice that could be screened for physiologically important and tissue-specific deficits. Conditional PamcKO/cKO mice, with loxP sites flanking the 2 exons deleted in the global PamKO/KO mouse, were indistinguishable from wild-type mice. Eliminating Pam expression in excitatory forebrain neurons reduced anxiety-like behavior, increased locomotor responsiveness to cocaine, and improved thermoregulation in the cold. A number of amidated peptides play essential roles in each of these behaviors. Although atrial natriuretic peptide (ANP) is not amidated, Pam expression in the atrium exceeds levels in any other tissue. Eliminating Pam expression in cardiomyocytes increased anxiety-like behavior and improved thermoregulation. Atrial and serum levels of ANP fell sharply in PAM myosin heavy chain 6 conditional knockout mice, and RNA sequencing analysis identified changes in gene expression in pathways related to cardiac function. Use of this screening platform should facilitate the development of therapeutic approaches targeted to peptidergic pathways.

Ciliary and cytoskeletal functions of an ancient monooxygenase essential for bioactive amidated peptide synthesis.

Many secreted peptides used for cell-cell communication require conversion of a C-terminal glycine to an amide for bioactivity. This reaction is catalyzed only by the integral membrane protein peptidylglycine α-amidating monooxygenase (PAM). PAM has been highly conserved and is found throughout the metazoa; PAM-like sequences are also present in choanoflagellates, filastereans, unicellular and colonial chlorophyte green algae, dinoflagellates and haptophytes. Recent studies have revealed that in addition to playing a key role in peptidergic signaling, PAM also regulates ciliogenesis in vertebrates, planaria and chlorophyte algae, and is required for the stability of actin-based microvilli. Here we briefly introduce the basic principles involved in ciliogenesis, the sequential reactions catalyzed by PAM and the trafficking of PAM through the secretory and endocytic pathways. We then discuss the multi-faceted roles this enzyme plays in the formation and maintenance of cytoskeleton-based cellular protrusions and propose models for how PAM protein and amidating activity might contribute to ciliogenesis. Finally, we consider why some ciliated organisms lack PAM, and discuss the potential ramifications of ciliary localized PAM for the endocrine features commonly observed in patients with ciliopathies.

Sex-Specific Gene Expression in the Mouse Nucleus Accumbens Before and After Cocaine Exposure.

The nucleus accumbens plays a major role in the response of mammals to cocaine. In animal models and human studies, the addictive effects of cocaine and relapse probability have been shown to be greater in females. Sex-specific differential expression of key transcripts at baseline and after prolonged withdrawal could underlie these differences. To distinguish between these possibilities, gene expression was analyzed in four groups of mice (cycling females, ovariectomized females treated with estradiol or placebo, and males) 28 days after they had received seven daily injections of saline or cocaine. As expected, sensitization to the locomotor effects of cocaine was most pronounced in the ovariectomized mice receiving estradiol, was greater in cycling females than in males, and failed to occur in ovariectomized/placebo mice. After the 28-day withdrawal period, RNA prepared from the nucleus accumbens of the individual cocaine- or saline-injected mice was subjected to RNA sequencing analysis. Baseline expression of 3% of the nucleus accumbens transcripts differed in the cycling female mice compared with the male mice. Expression of a similar number of transcripts was altered by ovariectomy or was responsive to estradiol treatment. Nucleus accumbens transcripts differentially expressed in cycling female mice withdrawn from cocaine exhibited substantial overlap with those differentially expressed in cocaine-withdrawn male mice. A small set of transcripts were similarly affected by cocaine in the placebo- or estradiol-treated ovariectomized mice. Sex and hormonal status have profound effects on RNA expression in the nucleus accumbens of naive mice. Prolonged withdrawal from cocaine alters the expression of a much smaller number of common and sex hormone-specific transcripts.

Transcriptional Profiling of Non-injured Nociceptors After Spinal Cord Injury Reveals Diverse Molecular Changes.

Traumatic spinal cord injury (SCI) has devastating implications for patients, including a high predisposition for developing chronic pain distal to the site of injury. Chronic pain develops weeks to months after injury, consequently, patients are treated after irreparable changes have occurred. Nociceptors are central to chronic pain; however, the diversity of this cellular population presents challenges to understanding mechanisms and attributing pain modalities to specific cell types. To begin to address how peripheral sensory neurons below the injury level may contribute to the below-level pain reported by SCI patients, we examined SCI-induced changes in gene expression in lumbar dorsal root ganglia (DRG) below the site of injury. SCI was performed at the T10 vertebral level, with injury produced by a vessel clip with a closing pressure of 15 g for 1 min. Alterations in gene expression produce long-term sensory changes, therefore, we were interested in studying SCI-induced transcripts before the onset of chronic pain, which may trigger changes in downstream signaling pathways and ultimately facilitate the transmission of pain. To examine changes in the nociceptor subpopulation in DRG distal to the site of injury, we retrograde labeled sensory neurons projecting to the hairy hindpaw skin with fluorescent dye and collected the corresponding lumbar (L2-L6) DRG 4 days post-injury. Following dissociation, labeled neurons were purified by fluorescence-activated cell sorting (FACS). RNA was extracted from sorted sensory neurons of naïve, sham, or SCI mice and sequenced. Transcript abundances validated that the desired population of nociceptors were isolated. Cross-comparisons to data sets from similar studies confirmed, we were able to isolate our cells of interest and identify a unique pattern of gene expression within a subpopulation of neurons projecting to the hairy hindpaw skin. Differential gene expression analysis showed high expression levels and significant transcript changes 4 days post-injury in SCI cell populations relevant to the onset of chronic pain. Regulatory interrelationships predicted by pathway analysis implicated changes within the synaptogenesis signaling pathway as well as networks related to inflammatory signaling mechanisms, suggesting a role for synaptic plasticity and a correlation with pro-inflammatory signaling in the transition from acute to chronic pain.

A pH-sensitive luminal His-cluster promotes interaction of PAM with V-ATPase along the secretory and endocytic pathways of peptidergic cells.

The biosynthetic and endocytic pathways of secretory cells are characterized by progressive luminal acidification, a process which is crucial for posttranslational modifications and membrane trafficking. This progressive fall in luminal pH is mainly achieved by the vacuolar-type-H+ ATPase (V-ATPase). V-ATPases are large, evolutionarily ancient rotary proton pumps that consist of a peripheral V1 complex, which hydrolyzes ATP, and an integral membrane V0 complex, which transports protons from the cytosol into the lumen. Upon sensing the desired luminal pH, V-ATPase activity is regulated by reversible dissociation of the complex into its V1 and V0 components. Molecular details of how intraluminal pH is sensed and transmitted to the cytosol are not fully understood. Peptidylglycine α-amidating mono-oxygenase (PAM; EC 1.14.17.3), a secretory pathway membrane enzyme which shares similar topology with two V-ATPase accessory proteins (Ac45 and prorenin receptor), has a pH-sensitive luminal linker region. Immunofluorescence and sucrose gradient analysis of peptidergic cells (AtT-20) identified distinct subcellular compartments exhibiting spatial co-occurrence of PAM and V-ATPase. In vitro binding assays demonstrated direct binding of the cytosolic domain of PAM to V1H. Blue native PAGE identified heterogeneous high-molecular weight complexes of PAM and V-ATPase. A PAM-1 mutant (PAM-1/H3A) with altered pH sensitivity had diminished ability to form high-molecular weight complexes. In addition, V-ATPase assembly status was altered in PAM-1/H3A expressing cells. Our analysis of the secretory and endocytic pathways of peptidergic cells supports the hypothesis that PAM serves as a luminal pH-sensor, regulating V-ATPase action by altering its assembly status.

Kalirin/Trio Rho GDP/GTP exchange factors regulate proinsulin and insulin secretion.

Key features for progression to pancreatic ß-cell failure and disease are loss of glucose responsiveness and an increased ratio of secreted proinsulin to insulin. Proinsulin and insulin are stored in secretory granules (SGs) and the fine-tuning of hormone output requires signal mediated recruitment of select SG populations according to intracellular location and age. The GTPase Rac1 coordinates multiple signaling pathways that specify SG release and Rac1 activity is controlled in part by GDP/GTP exchange factors (GEFs). To explore the function of two large multidomain GEFs, Kalirin and Trio in ß-cells, we manipulated their Rac1-specific GEF1 domain activity by using small molecule inhibitors and by genetically ablating Kalirin. We examined age related secretory granule behavior employing radiolabeling protocols. Loss of Kalirin/Trio function attenuated radioactive proinsulin release by reducing constitutive-like secretion and exocytosis of 2-hour old granules. At later chase times or at steady state, Kalirin/Trio manipulations decreased glucose stimulated insulin output. Finally, use of a Rac1 FRET biosensor with cultured ß-cell lines, demonstrated that Kalirin/Trio GEF1 activity was required for normal rearrangement of Rac1 to the plasma membrane in response to glucose. Rac1 activation can be evoked by both glucose metabolism and signaling through the incretin glucagon-like peptide 1 (GLP-1) receptor. GLP-1 addition restored Rac1 localization/activity and insulin secretion in the absence of Kalirin, thereby assigning Kalirin's participation to stimulatory glucose signaling.