Sequences within the Cytoplasmic Domain of Gp180/Carboxypeptidase D Mediate Localization to the Trans-Golgi Network

Gp180, a duck protein that was proposed to be a cell surface receptor for duck hepatitis B virus, is the homolog of metallocarboxypeptidase D, a mammalian protein thought to function in the trans-Golgi network (TGN) in the processing of proteins that transit the secretory pathway. Both gp180 and mammalian metallocarboxypeptidase D are type I integral membrane proteins that contain a 58-residue cytosolic C-terminal tail that is highly conserved between duck and rat. To investigate the regions of the gp180 tail involved with TGN retention and intracellular trafficking, gp180 and various deletion and point mutations were expressed in the AtT-20 mouse pituitary corticotroph cell line. Full length gp180 is enriched in the TGN and also cycles to the cell surface. Truncation of the C-terminal 56 residues of the cytosolic tail eliminates the enrichment in the TGN and the retrieval from the cell surface. Truncation of 12–43 residues of the tail reduced retention in the TGN and greatly accelerated the turnover of the protein. In contrast, deletion of the C-terminal 45 residues, which truncates a potential YxxL-like sequence (FxxL), reduced the protein turnover and caused accumulation of the protein on the cell surface. A point mutation of the FxxL sequence to AxxL slowed internalization, showing that this element is important for retrieval from the cell surface. Mutation of a pair of casein kinase II sites within an acidic cluster showed that they are also important for trafficking. The present study demonstrates that multiple sequence elements within the cytoplasmic tail of gp180 participate in TGN localization.

Molecular Cloning and Characterization of a Radial Spoke Head Protein of Sea Urchin Sperm Axonemes: Involvement of the Protein in the Regulation of Sperm Motility

Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40°C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI = 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form α-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.

Vesicle-associated Membrane Protein 4 is Implicated in Trans-Golgi Network Vesicle Trafficking

The trans-Golgi network (TGN) plays a pivotal role in directing proteins in the secretory pathway to the appropriate cellular destination. VAMP4, a recently discovered member of the vesicle-associated membrane protein (VAMP) family of trafficking proteins, has been suggested to play a role in mediating TGN trafficking. To better understand the function of VAMP4, we examined its precise subcellular distribution. Indirect immunofluorescence and electron microscopy revealed that the majority of VAMP4 localized to tubular and vesicular membranes of the TGN, which were in part coated with clathrin. In these compartments, VAMP4 was found to colocalize with the putative TGN-trafficking protein syntaxin 6. Additional labeling was also present on clathrin-coated and noncoated vesicles, on endosomes and the medial and trans side of the Golgi complex, as well as on immature secretory granules in PC12 cells. Immunoprecipitation of VAMP4 from rat brain detergent extracts revealed that VAMP4 exists in a complex containing syntaxin 6. Converging lines of evidence implicate a role for VAMP4 in TGN-to-endosome transport.

Two Components of Actin-based Retrograde Flow in Sea Urchin Coelomocytes

Sea urchin coelomocytes represent an excellent experimental model system for studying retrograde flow. Their extreme flatness allows for excellent microscopic visualization. Their discoid shape provides a radially symmetric geometry, which simplifies analysis of the flow pattern. Finally, the nonmotile nature of the cells allows for the retrograde flow to be analyzed in the absence of cell translocation. In this study we have begun an analysis of the retrograde flow mechanism by characterizing its kinetic and structural properties. The supramolecular organization of actin and myosin II was investigated using light and electron microscopic methods. Light microscopic immunolocalization was performed with anti-actin and anti-sea urchin egg myosin II antibodies, whereas transmission electron microscopy was performed on platinum replicas of critical point-dried and rotary-shadowed cytoskeletons. Coelomocytes contain a dense cortical actin network, which feeds into an extensive array of radial bundles in the interior. These actin bundles terminate in a perinuclear region, which contains a ring of myosin II bipolar minifilaments. Retrograde flow was arrested either by interfering with actin polymerization or by inhibiting myosin II function, but the pathway by which the flow was blocked was different for the two kinds of inhibitory treatments. Inhibition of actin polymerization with cytochalasin D caused the actin cytoskeleton to separate from the cell margin and undergo a finite retrograde retraction. In contrast, inhibition of myosin II function either with the wide-spectrum protein kinase inhibitor staurosporine or the myosin light chain kinase–specific inhibitor KT5926 stopped flow in the cell center, whereas normal retrograde flow continued at the cell periphery. These differential results suggest that the mechanism of retrograde flow has two, spatially segregated components. We propose a “push–pull” mechanism in which actin polymerization drives flow at the cell periphery, whereas myosin II provides the tension on the actin cytoskeleton necessary for flow in the cell interior.

The Yeast GRD20 Gene Is Required for Protein Sorting in the trans-Golgi Network/Endosomal System and for Polarization of the Actin Cytoskeleton

The proper localization of resident membrane proteins to the trans-Golgi network (TGN) involves mechanisms for both TGN retention and retrieval from post-TGN compartments. In this study we report identification of a new gene, GRD20, involved in protein sorting in the TGN/endosomal system of Saccharomyces cerevisiae. A strain carrying a transposon insertion allele of GRD20 exhibited rapid vacuolar degradation of the resident TGN endoprotease Kex2p and aberrantly secreted ∼50% of the soluble vacuolar hydrolase carboxypeptidase Y. The Kex2p mislocalization and carboxypeptidase Y missorting phenotypes were exhibited rapidly after loss of Grd20p function in grd20 temperature-sensitive mutant strains, indicating that Grd20p plays a direct role in these processes. Surprisingly, little if any vacuolar degradation was observed for the TGN membrane proteins A-ALP and Vps10p, underscoring a difference in trafficking patterns for these proteins compared with that of Kex2p. A grd20 null mutant strain exhibited extremely slow growth and a defect in polarization of the actin cytoskeleton, and these two phenotypes were invariably linked in a collection of randomly mutagenized grd20 alleles. GRD20 encodes a hydrophilic protein that partially associates with the TGN. The discovery of GRD20 suggests a link between the cytoskeleton and function of the yeast TGN.

Molecular basis for a link between complement and the vascular complications of diabetes

Activated terminal complement proteins C5b to C9 form the membrane attack complex (MAC) pore. Insertion of the MAC into endothelial cell membranes causes the release of growth factors that stimulate tissue growth and proliferation. The complement regulatory membrane protein CD59 restricts MAC formation. Because increased cell proliferation characterizes the major chronic vascular complications of human diabetes and because increased glucose levels in diabetes cause protein glycation and impairment of protein function, we investigated whether glycation could inhibit CD59. Glycation-inactivation of CD59 would cause increased MAC deposition and MAC-stimulated cell proliferation. Here, we report that (i) human CD59 is glycated in vivo, (ii) glycated human CD59 loses its MAC-inhibitory function, and (iii) inactivation of CD59 increases MAC-induced growth factor release from endothelial cells. We demonstrate by site-directed mutagenesis that residues K41 and H44 form a preferential glycation motif in human CD59. The presence of this glycation motif in human CD59, but not in CD59 of other species, may help explain the distinct propensity of humans to develop vascular proliferative complications of diabetes.

A structural basis for integrin activation by the cytoplasmic tail of the αIIb-subunit

A key step in the activation of heterodimeric integrin adhesion receptors is the transmission of an agonist-induced cellular signal from the short α- and/or β-cytoplasmic tails to the extracellular domains of the receptor. The structural details of how the cytoplasmic tails mediate such an inside-out signaling process remain unclear. We report herein the NMR structures of a membrane-anchored cytoplasmic tail of the αIIb-subunit and of a mutant αIIb-cytoplasmic tail that renders platelet integrin αIIbβ3 constitutively active. The structure of the wild-type αIIb-cytoplasmic tail reveals a “closed” conformation where the highly conserved N-terminal membrane-proximal region forms an α-helix followed by a turn, and the acidic C-terminal loop interacts with the N-terminal helix. The structure of the active mutant is significantly different, having an “open” conformation where the interactions between the N-terminal helix and C-terminal region are abolished. Consistent with these structural differences, the two peptides differ in function: the wild-type peptide suppressed αIIbβ3 activation, whereas the mutant peptide did not. These results provide an atomic explanation for extensive biochemical/mutational data and support a conformation-based “on/off switch” model for integrin activation.

Requirement of phosphatidylglycerol for photosynthetic function in thylakoid membranes

To investigate the role of phosphatidylglycerol (PG) in photosynthesis, we constructed a mutant defective in the CDP-diacylglycerol synthase gene from a cyanobacterium, Synechocystis sp. PCC6803. The mutant, designated as SNC1, required PG supplementation for growth. Growth was repressed in PG-free medium concomitantly with the decrease in cellular content of PG. These results indicate that PG is essential, and that SNC1 is defective in PG synthesis. Decrease in PG content was accompanied by a reduction in the cellular content of chlorophyll, but with little effect on the contents of phycobilisome pigments, which showed that levels of chlorophyll–protein complexes decreased without alteration of those of phycobilisomes. Regardless of the decrease in the PG content, CO2-dependent photosynthesis by SNC1 was similar to that by the wild type on a chlorophyll basis, but consequently became lower on a cell basis. Simultaneously, the ratio of oxygen evolution of photosystem II (PSII) measured with p-benzoquinone to that of CO2-dependent photosynthesis, which ranged between 1.3 and 1.7 in the wild type. However, it was decreased in SNC1 from 1.3 to 0.4 during the early growth phase where chlorophyll content and CO2-dependent photosynthesis were little affected, and then finally to 0.1, suggesting that PSII first lost its ability to reduce p-benzoquinone and then decreased in its level and actual activity. These results indicate that PG contributes to the accumulation of chlorophyll–protein complexes in thylakoid membranes, and also to normal functioning of PSII.

Molecular basis of lutropin recognition by the mannose/GalNAc-4-SO4 receptor

The circulatory half-life of the glycoprotein hormone lutropin (LH) is precisely regulated by the mannose (Man)/GalNAc-4-SO4 receptor expressed in hepatic endothelial cells. Rapid clearance from the circulation contributes to the episodic rise and fall of LH levels that is essential for maximal stimulation of the G protein-coupled LH receptor. We have defined two molecular forms of the Man/GalNAc-4-SO4 receptor that differ in ligand specificity, cell and tissue expression, and function. The form expressed by hepatic endothelial cells binds GalNAc-4-SO4-bearing ligands and regulates hormone circulatory half-life, whereas the form expressed by macrophages binds Man-bearing ligands and may play a role in innate immunity. We demonstrate that the GalNAc-4-SO4-specific form in hepatic endothelial cells is dimeric whereas the Man-specific form in lung macrophages is monomeric, accounting for the different ligand specificities of the receptor expressed in these tissues. Two cysteine-rich domains, each of which binds a single GalNAc-4-SO4, are required to form stable complexes with LH. The kinetics of LH binding by the GalNAc-4-SO4-specific form of the receptor in conjunction with its rate of internalization from the cell surface make it likely that only two of the four terminal GalNAc-4-SO4 moieties present on native LH are engaged before receptor internalization. As a result, the rate of hormone clearance will remain constant over a wide range of LH concentrations and will not be sensitive to variations in the number of terminal GalNAc-4-SO4 moieties as long as two or more are present on multiple oligosaccharides.

Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation

Wilson disease is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase. To elucidate the function of the Wilson protein, wild-type and mutant Wilson cDNAs were expressed in a Menkes copper transporter-deficient mottled fibroblast cell line defective in copper export. Expression of the wild-type cDNA demonstrated trans-Golgi network localization and copper-dependent trafficking of the Wilson protein identical to previous observations for the endogenously expressed protein in hepatocytes. Furthermore, expression of the Wilson cDNA rescued the mottled phenotype as evidenced by a reduction in copper accumulation and restoration of cell viability. In contrast, expression of an H1069Q mutant Wilson cDNA did not rescue the mottled phenotype, and immunofluorescence studies showed that this mutant Wilson protein was localized in the endoplasmic reticulum. Consistent with these findings, pulse–chase analysis demonstrated a 5-fold decrease in the half-life of the H1069Q mutant as compared with the wild-type protein. Maintenance of these transfected cell lines at 28°C resulted in localization of the H1069Q protein in the trans-Golgi network, suggesting that a temperature-sensitive defect in protein folding followed by degradation constitutes the molecular basis of Wilson disease in patients harboring the H1069Q mutation. Taken together, these studies describe a tractable expression system for elucidating the function and localization of the copper-transporting ATPases in mammalian cells and provide compelling evidence that the Wilson protein can functionally substitute for the Menkes protein, supporting the concept that these proteins use common biochemical mechanisms to effect cellular copper homeostasis.

Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function

During the aging process, mammals lose up to a third of their skeletal muscle mass and strength. Although the mechanisms underlying this loss are not entirely understood, we attempted to moderate the loss by increasing the regenerative capacity of muscle. This involved the injection of a recombinant adeno-associated virus directing overexpression of insulin-like growth factor I (IGF-I) in differentiated muscle fibers. We demonstrate that the IGF-I expression promotes an average increase of 15% in muscle mass and a 14% increase in strength in young adult mice, and remarkably, prevents aging-related muscle changes in old adult mice, resulting in a 27% increase in strength as compared with uninjected old muscles. Muscle mass and fiber type distributions were maintained at levels similar to those in young adults. We propose that these effects are primarily due to stimulation of muscle regeneration via the activation of satellite cells by IGF-I. This supports the hypothesis that the primary cause of aging-related impairment of muscle function is a cumulative failure to repair damage sustained during muscle utilization. Our results suggest that gene transfer of IGF-I into muscle could form the basis of a human gene therapy for preventing the loss of muscle function associated with aging and may be of benefit in diseases where the rate of damage to skeletal muscle is accelerated.

Multi-responsiveness of single anterior pituitary cells to hypothalamic-releasing hormones: A cellular basis for paradoxical secretion

The classic view for hypothalamic regulation of anterior pituitary (AP) hormone secretion holds that release of each AP hormone is controlled specifically by a corresponding hypothalamic-releasing hormone (HRH). In this scenario, binding of a given HRH (thyrotropin-, growth hormone-, corticotropin-, and luteinizing hormone-releasing hormones) to specific receptors in its target cell increases the concentration of cytosolic Ca2+ ([Ca2+]i), thereby selectively stimulating the release of the appropriate hormone. However, “paradoxical” responses of AP cells to the four well-established HRHs have been observed repeatedly with both in vivo and in vitro systems, raising the possibility of functional overlap between the different AP cell types. To explore this possibility, we evaluated the effects of HRHs on [Ca2+]i in single AP cells identified immunocytochemically by the hormone they stored. We found that each of the five major AP cell types contained discrete subpopulations that were able to respond to several HRHs. The relative abundance of these multi-responsive cells was 59% for lactotropes, 33% for thyrotropes, and in the range of 47–55% for gonadotropes, corticotropes, and somatotropes. Analysis of prolactin release from single living cells revealed that each of the four HRHs tested were able to induce hormone release from a discrete lactotrope subpopulation, the size of which corresponded closely to that in which [Ca2+]i changes were induced by the same secretagogues. When viewed as a whole, our diverse functional measurements of multi-responsiveness suggest that hypothalamic control of pituitary function is more complicated than previously envisioned. Moreover, they provide a cellular basis for the so-called “paradoxical” behavior of pituitary cells to hypothalamic hypophysiotropic agents.

The Ath5 proneural genes function upstream of Brn3 POU domain transcription factor genes to promote retinal ganglion cell development

During retinogenesis, the Xenopus basic helix–loop–helix transcription factor Xath5 has been shown to promote a ganglion cell fate. In the developing mouse and chicken retinas, gene targeting and overexpression studies have demonstrated critical roles for the Brn3 POU domain transcription factor genes in the promotion of ganglion cell differentiation. However, the genetic relationship between Ath5 and Brn3 genes is unknown. To understand the genetic regulatory network(s) that controls retinal ganglion cell development, we analyzed the relationship between Ath5 and Brn3 genes by using a gain-of-function approach in the chicken embryo. We found that during retinogenesis, the chicken Ath5 gene (Cath5) is expressed in retinal progenitors and in differentiating ganglion cells but is absent in terminally differentiated ganglion cells. Forced expression of both Cath5 and the mouse Ath5 gene (Math5) in retinal progenitors activates the expression of cBrn3c following central-to-peripheral and temporal-to-nasal gradients. As a result, similar to the Xath5 protein, both Cath5 and Math5 proteins have the ability to promote the development of ganglion cells. Moreover, we found that forced expression of all three Brn3 genes also can stimulate the expression of cBrn3c. We further found that Ath5 and Brn3 proteins are capable of transactivating a Brn3b promoter. Thus, these data suggest that the expression of cBrn3c in the chicken and Brn3b in the mouse is initially activated by Ath5 factors in newly generated ganglion cells and later maintained by a feedback loop of Brn3 factors in the differentiated ganglion cells.

Defective trafficking and function of KATP channels caused by a sulfonylurea receptor 1 mutation associated with persistent hyperinsulinemic hypoglycemia of infancy

The ATP-sensitive potassium channel (KATP) regulates insulin secretion in pancreatic β cells. Loss of functional KATP channels because of mutations in either the SUR1 or Kir6.2 channel subunit causes persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We investigated the molecular mechanism by which a single phenylalanine deletion in SUR1 (ΔF1388) causes PHHI. Previous studies have shown that coexpression of ΔF1388 SUR1 with Kir6.2 results in no channel activity. We demonstrate here that the lack of functional expression is due to failure of the mutant channel to traffic to the cell surface. Trafficking of KATP channels requires that the endoplasmic reticulum-retention signal, RKR, present in both SUR1 and Kir6.2, be shielded during channel assembly. To ask whether ΔF1388 SUR1 forms functional channels with Kir6.2, we inactivated the RKR signal in ΔF1388 SUR1 by mutation to AAA (ΔF1388 SUR1AAA). Inactivation of similar endoplasmic reticulum-retention signals in the cystic fibrosis transmembrane conductance regulator has been shown to partially overcome the trafficking defect of a cystic fibrosis transmembrane conductance regulator mutation, ΔF508. We found that coexpression of ΔF1388 SUR1AAA with Kir6.2 led to partial surface expression of the mutant channel. Moreover, mutant channels were active. Compared with wild-type channels, the mutant channels have reduced ATP sensitivity and do not respond to stimulation by MgADP or diazoxide. The RKR → AAA mutation alone has no effect on channel properties. Our results establish defective trafficking of KATP channels as a molecular basis of PHHI and show that F1388 in SUR1 is critical for normal trafficking and function of KATP channels.

Structural basis for fibroblast growth factor receptor 2 activation in Apert syndrome

Apert syndrome (AS) is characterized by craniosynostosis (premature fusion of cranial sutures) and severe syndactyly of the hands and feet. Two activating mutations, Ser-252 → Trp and Pro-253 → Arg, in fibroblast growth factor receptor 2 (FGFR2) account for nearly all known cases of AS. To elucidate the mechanism by which these substitutions cause AS, we determined the crystal structures of these two FGFR2 mutants in complex with fibroblast growth factor 2 (FGF2) . These structures demonstrate that both mutations introduce additional interactions between FGFR2 and FGF2, thereby augmenting FGFR2–FGF2 affinity. Moreover, based on these structures and sequence alignment of the FGF family, we propose that the Pro-253 → Arg mutation will indiscriminately increase the affinity of FGFR2 toward any FGF. In contrast, the Ser-252 → Trp mutation will selectively enhance the affinity of FGFR2 toward a limited subset of FGFs. These predictions are consistent with previous biochemical data describing the effects of AS mutations on FGF binding. Alterations in FGFR2 ligand affinity and specificity may allow inappropriate autocrine or paracrine activation of FGFR2. Furthermore, the distinct gain-of-function interactions observed in each crystal structure provide a model to explain the phenotypic variability among AS patients.