Functional Differences of Invariant and Highly Conserved Residues in the Extracellular Domain of the Glycoprotein Hormone Receptors

Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal hormone-binding fragment of the human FSH receptor (FSHR) extracellular domain (ECD). Binding of the other human glycoprotein hormones to their cognate human receptors (luteinizing hormone receptor (LHR) and thyroid-stimulating hormone receptor (TSHR)) was expected to be similar. This study focuses on amino acid residues in β-strands 2 (Lys⁷⁴), 4 (Tyr¹²⁴, Asn¹²⁹, and Thr¹³⁰), and 5 (Asp¹⁵⁰ and Asp¹⁵³) of the FSHR ECD identified in the human FSH·FSHR ECD crystal structure as contact sites with the common glycoprotein hormone α-subunit, and on noncontact residues in β-strands 2 (Ser⁷⁸) and 8 (Asp²²⁴ and Ser²²⁶) as controls. These nine residues are either invariant or highly conserved in LHR and TSHR. Mutagenesis and functional characterization of these residues in all three human receptors allowed an assessment of their contribution to binding and receptor activation. Surprisingly, the six reported α-subunit contact residues of the FSHR ECD could be replaced without significant loss of FSH binding, while cAMP signaling potency was diminished significantly with several replacements. Comparative studies of the homologous residues in LHR and TSHR revealed both similarities and differences. The results for FSH/FSHR were analyzed on the basis of the crystal structure of the FSH·FSHR ECD complex, and comparative modeling was used to generate structures for domains, proteins, and complexes for which no structures were available. Although structural information of hormone-receptor interaction allowed the identification of hormone-receptor contact sites, functional analysis of each contact site was necessary to assess its contribution to hormone binding and receptor activation.     

Affinity binding of hamster oviductin to spermatozoa and its influence on in vitro fertilization

The aim of this study was to determine the influence of hamster oviductal glycoprotein (Oviductin) on in vitro gamete interaction. Oviductin was purified from the oviducts using lithium 3,5-diiodosalicylate, followed by phenol extraction. Immunocytochemistry using indirect fluorescence staining revealed that oviductin binds to the sperm anterior acrosomal region. The specific binding of oviductin resulted in inhibition of in vitro fertilization in studies using cumulus-free oocytes. The inhibitory effect was dependent on the concentration of oviductin and occurred in both ovarian and oviductal oocytes but not zona-free oocytes, indicating that sperm-zona interaction was interferred by oviduction. However, the inhibitory effect of oviductin sperm-zona interaction was reduced when cumulus-enclosed oocytes from ovaries and oviducts were used, indicating that the egg investment including cumulus oophorus has some effect on oviductin-sperm complex and maintaining the fertilizing ability. © Wiley-Liss, Inc.     

RhoG Protein Regulates Platelet Granule Secretion and Thrombus Formation in Mice

Rho GTPases such as Rac, RhoA, and Cdc42 are vital for normal platelet function, but the role of RhoG in platelets has not been studied. In other cells, RhoG orchestrates processes integral to platelet function, including actin cytoskeletal rearrangement and membrane trafficking. We therefore hypothesized that RhoG would play a critical role in platelets. Here, we show that RhoG is expressed in human and mouse platelets and is activated by both collagen-related peptide (CRP) and thrombin stimulation. We used RhoG^−/− mice to study the function of RhoG in platelets. Integrin activation and aggregation were reduced in RhoG^−/− platelets stimulated by CRP, but responses to thrombin were normal. The central defect in RhoG^−/− platelets was reduced secretion from α-granules, dense granules, and lysosomes following CRP stimulation. The integrin activation and aggregation defects could be rescued by ADP co-stimulation, indicating that they are a consequence of diminished dense granule secretion. Defective dense granule secretion in RhoG^−/− platelets limited recruitment of additional platelets to growing thrombi in flowing blood in vitro and translated into reduced thrombus formation in vivo. Interestingly, tail bleeding times were normal in RhoG^−/− mice, suggesting that the functions of RhoG in platelets are particularly relevant to thrombotic disorders.     

The recombinant equine LHβ subunit combines divergent intracellular traits of human LHβ and CGβ subunits

The pituitary LHβ and placental CGβ subunits are products of different genes in primates. The major structural difference between the two subunits is in the carboxy-terminal region, where the short carboxyl sequence of hLHβ is replaced by a longer O-glycosylated carboxy-terminal peptide in hCGβ. In association with this structural deviation, there are marked differences in the secretion kinetics and polarized routing of the two subunits. In equids, however, the CGβ and LHβ subunits are products of the same gene expressed in the placenta and pituitary (LHβ), and both contain a carboxy-terminal peptide. This unusual expression pattern intrigued us and led to our study of eLHβ subunit secretion by transfected Chinese hamster ovary and Madin–Darby canine kidney cells. In continuous labeling and pulse-chase experiments, the secretion of the eLHβ subunit from the transfected Chinese hamster ovary cells was inefficient (medium recovery of 16%–25%) and slow (t_1/2 > 6.5 hours). This indicated that, the secretion of the eLHβ subunit resembles that of hLHβ rather than hCGβ. In Madin–Darby canine kidney cells grown on Transwell filters, the eLHβ subunit was preferentially secreted from the apical side, similar to the hCGβ subunit secretory route (∼65% of the total protein secreted). Taken together, these data suggested that secretion of the eLHβ subunit integrates features of both hLHβ and hCGβ subunits. We propose that the evolution of this intracellular behavior may fulfill the physiological demands for biosynthesis of the LH and CG β-subunits in the pituitary and placenta, respectively.     

Trypanosoma congolense: Surface glycoproteins of two early bloodstream variants: II. Purification and partial chemical characterization

Two sequential variant-specific glycoproteins have been purified from two variants of Trypanosoma congolense expressed during a relapsing infection. Isolation of the two glycoproteins, termed VSG-1 and VSG-2, respectively, employed glycerol lysis followed by purification on concanavalin A, Sephadex G-25, and gradient-eluted DE-52 columns. Partially purified VSG proteins were immunologically cross-reactive, but highly purified VSGs showed no cross-reactivity under the conditions employed. Both VSG-1 and VSG-2 consisted of a triplet of polypeptides. Although each member of a triplet subset could be distinguished by isoelectric focusing, all three gave identical N-terminal amino acid sequences and nearly identical tryptic peptide maps. The members of the VSG-1 polypeptide subset differed from those of the VSG-2 subset both with regard to N-terminal amino acid sequence and in tryptic peptide map patterns. Comparison of N-terminal sequences of VSG-1 and VSG-2 did, however, show that the sequences could be aligned to give a modest degree of amino acid homology (27%). This alignment also produced a minimum in the number of two-base changes, suggesting that the observed homology is not a coincidence and that these two proteins may well have arisen by gene duplication followed by retention of multiple point mutations.     

Specific modification of gastric K+-stimulated ATPase activity by thimerosal

Treatment of hog gastric microsomes with the sulfhydryl reagent, thimerosal (ethylmercurithiosalicylate), produced differential effects on the K⁺-ATPase and the K⁺-stimulated p-nitrophenylphosphatase activities. For example, exposure to 2 mM thimerosal for 3 min severely reduced the activity of K⁺-stimulated ATPase, while K⁺-p-nitrophenylphosphatase activity was enhanced 2- to 3-fold. Higher concentration of thimerosal, or longer incubation times, also led to inhibition of K⁺-p-nitrophenylphosphatase. The activated state of p-nitrophenylphosphatase could be sustained by a 20-fold, or greater, dilution of treated membranes, and could be reversed by reduction of membrane SH groups by exogenous thiols. Significant activation of K⁺-p-nitrophenylphosphatase was not produced by p-chloromercuribenzene sulfonate, p-chloromercuribenzoate or mersalyl; however, ethyl mercuric chloride had qualitatively similar activity effects as thimerosal. Kinetics of K⁺-p-nitrophenylphosphatase for thimerosal-treated membranes were altered as follows: V increased; K_m for p-nitrophenylphosphate unchanged for K_a for K⁺ increased. ATP, which is a potent inhibitor of K⁺-p-nitrophenylphosphatase activity in native membranes (K_I ≈ 200 μM). These data suggest that there are multiple SH groups which differentially influence the gastric K⁺-stimulated ATPase activity. Defined treatments with thimerosal are interpreted as an uncoupling of the K⁺-stimulated phosphatase component of the enzyme (for which p-nitrophenylphosphatase is a presumed model reaction). Such differential modifications can be usefully applied to the study of partial reactions of the enzyme and their specific role in the related H⁺-transport reaction.     

Plasmodium falciparum: Assay in vitro for inhibitors of merozoite penetration of erythrocytes

Highly synchronous cultures of the erythrocytic stages of Plasmodium falciparum were used both to assay penetration of merozoites into human red blood cells, and to subsequently study the inhibitory effects of various substances on penetration. While several sugars exhibited no inhibitory effect, fucose, glucosamine-HCl, and N-acetyl glucosamine, when added to synchronous cultures at the schizont stage, inhibited invasion. On further testing fucose and glucosamine-HCl were found to be toxic to the intracellular growth and development of the parasite; only N-acetyl glucosamine had an inhibitory effect solely related to the inhibition of merozoite penetration. Glycophorin A, the major glycoprotein of the red blood cell surface, had no inhibitory effect at low concentrations, but had a slight effect at higher (500 μg/ml) levels.     

The phorbol ester TPA increases the affinity of exocytosis for calcium in ‘leaky’ adrenal medullary cells

Exposure of ‘leaky’ bovine adrenal medullary cells to the phorbol ester TPA causes a shift in the calcium-activation curve to lower calcium concentrations without altering the levels of secretion at the extremes of the activation curve. These results are consistent with a role for protein kinase C in exocytosis.     

Transient occurrence of lipoxygenase and glycoprotein gp49 in lipid bodies during fat mobilization in anise seedlings

The patterns of lipid-body proteins prepared at different stages of germination from seedlings of anise (Pimpinella ansium L.) were investigated. During the stage of fat mobilization, a set of proteins is synthesized de novo and transferred to existing lipid bodies. Analysis of the protein constituents, detected either by protein staining, concanavalin-A/peroxidase staining or immunoreaction on Western blots showed three distinct protein species: a lipoxygenase firmly integrated into the lipid bodies, a glycoprotein gp50 only extractable from the lipid bodies by treatment with sodium dodecyl sulfate and a glycoprotein gp49 solubilized by diethyl-ether treatment of lipid bodies. Both the lipoxygenase immunodetected on Western blots and gp49 stained with concanavalin-A/ peroxidase conjugate occurred transiently in the lipidbody fraction, reaching maximum concentrations between days 6 and 13 of germination. This behavior was in contrast to the decreasing level of the 18.4-kDa oleosin already present at the beginning of germination.Abbreviations gp glycoprotein - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate We would like to thank Christiane Bernshausen and Willi Jonghaus for kind technical assistance. This research was supported by a grant from the Deutsche Forschungsgemeinschaft. I.F. is a recipient of the Friedrich Ebert-Stiftung.     

Molecular motors and the Golgi complex: Staying put and moving through

The Golgi apparatus is a highly dynamic organelle through which nascent proteins released from the endoplasmic reticulum (ER) are trafficked. Proteins are post-translationally modified within the Golgi and subsequently packaged into carriers for transport to a variety of cellular destinations. This transit of proteins, as well as the maintenance of Golgi structure and position, is highly dependent upon the actin and microtubule cytoskeletons and their associated molecular motors. Here we review how motors contribute to the correct functioning of the Golgi in higher eukaryotes and discuss the secretory pathway as a model system for studying cooperation between motor proteins.