We study the cell biology of endocrine and neuroendocrine cells. Our focus is two-fold: (1) to investigate the mechanisms of biosynthesis and intracellular trafficking of peptide hormones and neuropeptides and their processing enzymes and (2) to uncover mechanisms involved in the regulation of dense-core secretory granule biogenesis. Our work has led to the discovery of novel molecular mechanisms of protein trafficking to the regulated secretory pathway and the identification of players and mechanisms that control secretory granule biogenesis and transport in endocrine and neuroendocrine cells and neurons. Using cell lines, primary cell cultures, and mouse models, our studies have provided a better understanding of diseases related to defects in hormone and neuropeptide targeting, synaptic transmission, obesity, and cholesterol deficiency.
Mechanism of sorting pro-neuropeptides and neurotrophins to the regulated secretory pathway
Cawley, Zhang, Lou, Loh; in collaboration with Baum, Chow, Lu, Snell
The intracellular sorting of pro-neuropeptides and neurotrophins to the regulated secretory pathway (RSP) is essential for processing, storing, and releasing active proteins and peptides in the neuroendocrine cell. We investigated the sorting of pro-opiomelanocortin (POMC, pro-ACTH/endorphin), proinsulin, and brain-derived neurotrophic factor (BDNF) to the RSP. We showed that, as a concentration step, these pro-proteins undergo homotypic oligomerization as they traverse the cell from the site of synthesis in the endoplasmic reticulum to the trans-Golgi network (TGN), where they are sorted into dense-core granules of the RSP for processing and secretion. Site-directed mutagenesis studies identified a consensus sorting motif consisting of two acidic residues, 12 to 15Å apart, which are exposed on the surface of these molecules, and two hydrophobic residues, at a distance of 5 to 7Å from the acidic residues, which are necessary for sorting to the RSP. While such a motif was found in BDNF, which is secreted in an activity-dependent manner, nerve growth factor (NGF), which is primarily secreted constitutively, was lacking one amino acid residue of the motif. Introduction of the missing residue by mutagenesis (Val20Glu) redirected NGF to the RSP, further confirming the importance of the sorting motif in targeting to the RSP. We identified membrane carboxypeptidase E (CPE) as an RSP sorting receptor that was specific for the sorting signal of POMC, proinsulin, and BDNF. To effect sorting to the RSP, the two acidic residues in the prohormone/pro-BDNF sorting motif specifically interact with R255 and K260, two basic residues of the sorting receptor CPE. Transfection of a mutant CPE with R255 and K260 that had been mutated to A in a CPE-null clone of Neuro2a cells caused missorting of co-transfected POMC to the constitutive pathway, indicating that the basic residues in the sorting domain of CPE interact with the acidic residues in the POMC sorting signal in vivo to effect sorting to the RSP. Transfection of a dominant negative CPE mutant into cells of the rat insulinoma line (INS cells) or depletion of CPE in these cells using siRNA caused partial missorting of proinsulin to the constitutive pathway, indicating an in vivo interaction of proinsulin and CPE to effect sorting. Furthermore, using a CPE knockout (KO) mouse model, we showed that BDNF was not sorted to the RSP but was secreted constitutively in cortical and hippocampal neurons of the CPE KO mice. Constitutive secretion of proinsulin from isolated pancreatic islets in the mouse model appeared to be elevated while plasma levels of proinsulin were significantly higher, indicating a role for CPE in the intracellular trafficking of proinsulin. Our studies provide evidence for a sorting signal–/receptor-mediated mechanism for targeting prohormones, pro-neuropeptides, and the neurotrophin BDNF to the RSP in neuro-endocrine cells and neurons.
In collaboration with Bruce Baum, we have used our knowledge of the sorting motif of hormones to engineer biologically active mutant hormones that are redirected to the constitutive pathway. We are currently expressing these mutant hormones in salivary glands for systemic secretion, with the ultimate aim of applying such technology to gene therapeutics. Recently, we succeeded in expressing a biologically active mutant growth hormone in salivary glands and partially redirecting it to the constitutive pathway for systemic secretion.
Cawley NX, Arnaoutova I, Yanik T, Lou H, Patel N, Loh YP. Techniques in neuropeptide processing, trafficking, and secretion. In: Gozes I, ed. Methods in Neurobiology. Humana Press, 2005 (in press).
Lou H, Kim S-K, Zaitsev E, Snell C, Lu B, Loh YP. Sorting and activity-dependent secretion of BDNF require interaction of motif I16D18I105E106 with the sorting receptor, carboxypeptidase E. Neuron 2005;45:245-255.
Michael D, Geng X, Cawley NX, Loh YP, Rhodes C, Drain P, Chow R. Fluorescent cargo proteins in pancreatic beta cells: design determines secretion kinetics at exocytosis. Biophys J 2004;87:103-105.
Wang J, Cawley NX, Voutetakis A, Rodriguez Y, Goldsmith C, Nieman L, Hoque S, Frank S, Snell C, Loh YP, Baum B. Partial re-direction of transgenic human growth hormone secretion from rat salivary glands. Hum Gene Ther 2005;16:571-583.
Obesity in humans resulting from intracellular missorting of mutant (Leu34Phe) pro-CART
Yanik, Loh; in collaboration with Kuhar, del Guidice
In another study, we investigated the sorting and processing of a mutant form of cocaine-amphetamine–regulated transcript (CART) found in a family of obese patients. CART is an anorectic brain peptide that has several physiological effects such as the inhibition of feeding and the regulation of energy expenditure and stress. CART acts downstream of leptin in the obesity-controlling signaling pathway. A mutant pro-CART (Leu34Phe) was found in a 10-year-old Italian boy who has been obese since two years of age. This missense Leu34Phe mutation, along with the phenotype of severe obesity, co-segregates in three generations of the boy’s maternal relatives, but his father was not obese. To investigate whether these patients have mature CART, we used Protein Chip technology (Ciphergen) to analyze serum from the obese boy and affected family members from three generations and from an unaffected sibling and two normal controls. All members of the family bearing the (Phe34Leu) mutation showed only pro- and intermediate CART in their circulation, but no mature CART. In contrast, normal humans and the unaffected sibling showed significant amounts of circulating mature CART. To determine the cellular basis for the lack of mature CART in the obese patients, we investigated the trafficking and processing of mutant CART by transfecting wild-type (WT) or mutant (Leu34Phe) CART into AtT-20 cells. While pro-CART was substantially processed to active CART, mutant pro-CART was only minimally processed to yield an intermediate form in these cells. Furthermore, WT CART was secreted in a regulated manner with high potassium stimulation, but mutant pro-CART/CART exhibited high basal release and no significant stimulated secretion. Immunocytochemical studies revealed that immunoreactive WT CART was primarily co-localized in punctate granules with POMC, a granule marker, in the processes of AtT-20 cells. However, about 50 percent of the cells showed no punctate staining of immunoreactive mutant CART co-localized with POMC in the cell processes. The results indicate that mutant pro-CART was partially missorted and secreted via the constitutive pathway. The poor processing of mutant pro-CART is likely attributable to the missorting to the constitutive pathway, which lacks the appropriate processing enzymes. Thus, the missorting and lack of processing of mutant CART (Leu34Phe) provides a molecular basis for the obese phenotype in the subjects.
Yanik T, Dominguez G, Kuhar MJ, del Guidice EM, Loh YP. The Leu34Phe ProCART mutation leads to CART deficiency. A possible cause for obesity in humans. Endocrinology 2005 [Epub ahead of print].
Deficits in synaptic transmission and synaptic vesicle trafficking exhibited by the CPE knockout mouse
Cawley, Park, Yanik, Loh; in collaboration with Craft, Hill, Wetsel
We generated a carboxypeptidase E knockout (CPE KO) mouse by deleting exons 4 and 5 from the CPE gene and then characterized its phenotype. The KO mice became obese by 10 to 12 weeks of age and weighed 60 to 80 grams by 40 weeks. At this age, body fat content was more than double that of the WT controls. The null animals consumed more food overall, were less physically active during the light phase of the light-dark cycle, and burned fewer calories as fat than WT littermates. Fasting levels of glucose and insulin-like immunoreactivity (IR) in plasma were elevated in both male and female KO mice at about 20 weeks; males recovered from this state by 32 weeks, but females did not. Nevertheless, at this time, IR was 50 to 100 times higher than that of the WT controls. We identified the plasma insulin-like IR material as primarily proinsulin. The KO mice showed impaired glucose clearance and were insulin-resistant. They also evidenced high levels of leptin in plasma but showed no circulating, fully processed CART, a peptide responsive to leptin-induced feedback inhibition of feeding. In addition to the obesity and diabetes phenotypes, the KO mice were subfertile and showed deficits in GnRH processing in the hypothalamus.
Behavioral analyses revealed that KO animals had diminished reactivity to stimuli as well as reduced muscle strength, coordination and visual placing, and toe-pinch reflexes. Moreover, they showed delayed learning in the water-maze test. The mice also exhibited abnormal neurotransmission from the photoreceptors to the inner retina, with a loss of the b wave in their retinogram. We obtained similar results in a mutant mouse (Cpefat/fat) bearing a substitution mutation in residue Ser202Pro of CPE. Immunocytochemical studies in the latter mice revealed a lack of CPE in the outer plexiform layer but an accumulation in the outer nuclear layer where, unlike in normal mice, the cell bodies of the photoreceptor cells are localized. Furthermore, electron microscopy studies indicated significantly fewer synaptic vesicles in the spherules (synaptic butons), suggesting a defect in transport of synaptic vesicles carrying CPE. Thus, CPE may play a role in transport of synaptic vesicles. Indeed, in pull-down experiments, we recently found that the cytoplasmic tail of CPE interacts with snapin, SNAP25, and microtubule-based motors. Overexpression of the CPE tail in AtT-20 cells inhibited transport of CPE-containing regulated secretory pathway granules to the cell processes for release, thereby pointing to a novel role of CPE in vesicle transport.
Cawley NX, Zhou J, Hill J, Abebe D, Romboz S, Yanik T, Rodriguiz Y, Wetsel W, Loh YP. The carboxypeptidase E knockout mouse exhibits endocrinological and behavioral deficits. Endocrinology 2004;145:5807-5819.
Zhu X, Wu K, Rife L, Cawley NX, Brown B, Adams T, Teofilo K, Lillo C, Williams D, Loh YP, Craft C. Carboxypeptidase E is essential for normal synaptic transmission from photoreceptors to the inner retina. J. Neurochem 2005;95:1351-1362.
Role of cholesterol in prohormone-processing enzyme sorting and granule biogenesis
Gondre-Lewis, Lou, Loh; in collaboration with Birch, Parsegian, Porter
Our recent studies showed that the prohormone- and neuropeptide-processing enzymes carboxypeptidase E and prohormone convertases 1/3 and 2 (PC1 and PC2) are transmembrane proteins with an atypical membrane-spanning domain at the C-terminus. In neuroendocrine cells, they are sorted at the TGN into granules of the RSP by a novel mechanism involving transmembrane association of their C-terminal domain into cholesterol-glycosphingolipid–rich microdomains known as lipid rafts. Removal of cholesterol from secretory granule membranes resulted in the inability of CPE, an RSP sorting receptor, to bind to cargo; in fact, cholesterol depletion by treatment of cells with lovastatin prevented the sorting of CPE to the RSP. Furthermore, removal of the transmembrane domain of PC1/3 led to the missorting of the enzyme to the constitutive pathway. Thus, membrane association with cholesterol-rich lipid rafts is essential for sorting of the prohormone-processing enzymes to the TGN.
To assess the importance of cholesterol in secretory granule biogenesis and in packaging of granule content in vivo, we analyzed vesicles in the pancreas of cholesterol-deficient mouse models of Smith-Lemli Opitz syndrome (SLOS) and lathosterolosis (Sc5d–/–). SLOS and lathosterolosis are human disorders caused by, respectively, defective 7-dehydrocholesterol reductase and lathosterol 5-desaturase, enzymes necessary for the final steps of cholesterol synthesis. Morphological analysis by light and electron microscopy of neonatal pancreas zymogen granules in the mouse model showed markedly fewer granules in both SLOS and Sc5d–/– than in control mice. Of the granules present in SLOS and Sc5d–/–animals, most were of an immature phenotype, appearing as partially formed spheres. Sc5d–/– exocrine pancreas lacking granules was filled with rough ER ribbons and ribosomal structures, indicating an inability to package materials into membrane-bound structures. Furthermore, in primary cultures of cholesterol-deficient secretory cells in the exocrine pancreas, protein synthesis and regulated secretion were impaired as compared with control cells. We hypothesize that the defect in granule biogenesis and maturation is attributable to different physical contributions of sterols to membrane curvature. Indeed, biophysical studies indicate that 7-dehydro-cholesterol and lathosterol have a lower binding rigidity than cholesterol and therefore exhibit a poorer ability to form curvature. Thus, genetic inhibition of cholesterol synthesis in SLOS and Sc5d–/– impairs regulated secretory pathway granule biogenesis and maturation, leading to deficits in the secretory function in the exocrine pancreas and possibly in the endocrine and nervous systems.
Regulation of secretory granule biogenesis by chromogranin A
Kim, Arnaoutova, Zhang, Loh; in collaboration with Pickel, Wu
Formation of large dense-core granules (LDCGs) at the TGN is essential for the regulated secretion of hormones and neuropeptides from neuroendocrine cells. Our recent studies uncovered an on/off switch, chromogranin A (CgA), that controls the formation of LDCGs in neuroendocrine cells. Using antisense technology, we demonstrated that depletion of CgA in rat PC12 cells results in the loss of LDCGs and the regulated secretion and degradation of granule proteins, including CgB and synaptotagmin. Overexpression of bovine CgA in the cells rescued the wild-type phenotype. In 6T3, a mutant endocrine cell line lacking CgA, LDCGs, and regulated hormone secretion, transfection of CgA resulted in the rescue of granule biogenesis and the regulated secretory pathway. The importance of CgA in LDCG biogenesis was evident not only in cell lines but also in vivo. In an antisense mRNA transgenic mouse model deficient in CgA, which we generated in collaboration with James Pickel, we observed severe aberrant granule formation both quantitatively and qualitatively in the adrenal medulla of the mice, with a correlation between the amount of depletion of CgA and the reduction in secretory granule biogenesis. A reduction in granule proteins accompanied the reduction in secretory granule biogenesis in the adrenal medulla of the CgA-deficient transgenic animals and in 6T3 cells lacking CgA. Using 6T3 cells lacking secretory granules as a model, we showed that the reduced granule protein levels were attributable to degradation occurring at the Golgi apparatus. Thus, we proposed that regulation of the stability of granule proteins at the Golgi apparatus by CgA is a focal point for control of granule biogenesis in neuroendocrine cells. To this end, we recently found a protease inhibitor in the Golgi that is transcriptionally activated by CgA. The inhibitor is upregulated in cells actively forming LDCGs but downregulated in cells that minimally express CgA and show low levels of LDCG biogenesis. Moreover, transfection of the protease inhibitor into 6T3 cells lacking CgA prevented LDCG protein degradation and rescued granule biogenesis. Furthermore, downregulation of expression of the protease inhibitor by antisense RNA in 6T3 cells transfected with CgA resulted in enhanced degradation of granule proteins and decreased secretory granule formation. Thus, we have uncovered a novel mechanism whereby CgA regulates large dense-core granule biogenesis by transcriptionally activating a protease inhibitor that stabilizes granule proteins necessary for LDCG biogenesis.
Kim T, Gondre-Lewis M, Arnaoutova I, Cawley NX, Loh YP. Neurosecretory protein trafficking and dense-core granule biogenesis in neuroendocrine cells. In: Lajtha A, Banik N, eds. Handbook of Neurochemistry and Molecular Neurobiology, 3rd ed., Vol. 7, Neural Proteins: Metabolism and Function. Kluwer-Academic-Pleum, 2005 (in press).
Kim T, Loh YP. Chromogranin A: the surprise link between granule biogenesis and hypertension. J Clin Invest 2005;115:1711-1713.
Kim T, Zhang C-F, Sun Z, Wu H, Loh YP. Chromogranin A deficiency in transgenic mice leads to aberrant chromaffin granule biogenesis. J Neurosci 2005;25:6958-6961.
Role of Aquaporin 1 in hormone secretion and secretory granule biogenesis
Arnaoutova, Kim, Loh
Recently, we found that the water channel protein Aquaporin 1 (AQP1) is localized in secretory granules and the plasma membrane of endocrine cells. Moreover, when we transfected 6T3 cells lacking CgA with CgA, which rescues granule biogenesis in these cells, we observed a significant increase in the expression of AQP1 mRNA and protein. To investigate the role of AQP1 in hormone sequestration and granule biogenesis, we stably transfected AtT-20 cells with an antisense construct of AQP1 to downregulate expression of the protein. AQP1-deficient AtT-20 cells showed a dramatic reduction of secretory granules (see Figure 23.1). Pulse-chase studies demonstrated a defect in regulated secretion of the endogenous ACTH hormone as well as increased degradation of newly synthesized granule proteins such as POMC and CPE at the Golgi apparatus. However, the deficiency of AQP1 in the AtT-20 cells did not affect the transcription and translation of these proteins. Thus, AQP1 appears to be critical for the mechanism of secretory granule exocytosis and hormone sequestration. A defect in secretion leads to a feedback to downregulate secretory granule biogenesis. Interestingly, the regulation of the downregulation of granule biogenesis appears to occur at the post-translational level through enhanced degradation of newly synthesized granule proteins.
Collaborators
Bruce Baum, DMD, Gene Therapy and Therapeutics Branch, NIDCR, Bethesda, MD
Nigel Birch, PhD, University of Auckland, Auckland, New Zealand
Robert Chow, PhD, University of Southern California, Los Angeles, CA
Cheryl Craft, PhD, University of Southern California, Los Angeles, CA
Emanuele del Guidice, MD, Seconda Università di Napoli, Naples, Italy
Joanna Hill, PhD, Laboratory of Developmental Neurobiology, NICHD, Bethesda, MD
Michael Kuhar, PhD, Yerkes National Primate Center, Emory University, Atlanta, GA
Bai Lu, PhD, Laboratory of Cellular and Synaptic Neurophysiology, NICHD, Bethesda, MD
Adrian Parsegian, PhD, Laboratory of Physical and Structural Biology, NICHD, Bethesda, MD
James Pickel, PhD, Laboratory of Genetics, NIMH, Bethesda, MD
Forbes Porter, MD, PhD, Heritable Disorders Branch, NICHD, Bethesda, MD
Chris Snell, PhD, Medivir UK Ltd., Chesterford, UK
William Wetsel, PhD, Duke University, Durham, NC
Heling Wu, PhD, Peking University, Beijing, China
For further information, contact lohp@mail.nih.gov.