Carbachol-induced desensitization of PLC-beta pathway in rat myometrium: downregulation of Gqalpha /G11alpha

In the estrogen-treated rat myometrium, carbachol increased thegeneration of inositol phosphates by stimulating the muscarinic receptor-G_q/G₁₁-phospholipaseC-3 (PLC-3) cascade. Exposure to carbachol resulted in a rapidand specific (homologous) attenuation of the subsequent muscarinicresponses in terms of inositol phosphate production, PLC-3translocation to membrane, and contraction. Refractoriness wasaccompanied by a reduction of membrane muscarinic binding sites and anuncoupled state of residual receptors. Protein kinase C (PKC) alteredthe functionality of muscarinic receptors and contributed to theinitial period of desensitization. A delayed phase of the muscarinicrefractoriness was PKC independent and was associated with adownregulation ofG_q/G₁₁.Atropine failed to induce desensitization as well asG_q/G₁₁downregulation, indicating that both events involve active occupancy ofthe receptor. Prolonged exposure toAlF₄ reduced subsequent AlF₄ as well as carbachol-mediatedinositol phosphate responses and similarly induced downregulation ofG_q/G₁₁. Data suggest that a decrease in the level ofG_q/G₁₁is subsequent to its activation and may account forheterologous desensitization.

     

Sialylation of intestinal microvillar membranes in newborn, sucking and weaned pigs

Affinity cytochemistry and biochemistry revealed distinctivetemporal changes in the expression of sialylated and compositionallyrelated membrane glycoconjugates in the pig small intestinebetween birth and weaning. The expression of membrane NeuAc2,6moieties, recognized by Sambucus nigra agglutinin-1, was highin newborn pigs, declined slightly during sucking and was verylow in weaned animals. Conversely, the expression of membraneNeuAc2r3 moieties, recognized by Maackia amurensis agglutinin-2,was low at birth but higher in sucking and weaned animals. Histobloodgroup O- and A-antigen expression was first detected in a minorityof sucking pigs, but was evident in all weaned pigs examined.Lactase glycoforms were isolated from solubilized microvillarmembranes of newborn and weaned pigs. The newborn (predominantly2,6-sialylated) and weaned (predominantly 1,2-fucosylated) glycoformsexhibited similar specific activity, indicating that postnatallactase decline in the pig intestine is unrelated to temporalchanges in membrane sialylation and fucosylation. fucosylation lactase lectins intestine sialylation     

Cytoskeletal disruption and small heat shock protein translocation immediately after lengthening contractions

The purposes of this study were to determine whether, immediately after lengthening contractions, 1) levels of specific force-transmitting cytoskeletal elements are reduced in skeletal muscle cells and 2) cytosolic small heat shock proteins (HSPs) translocate to structures prone to disruption. Western blot analysis demonstrated decreased concentrations of z-disk proteins -actinin and plectin and membrane scaffolding proteins dystrophin and -spectrin in muscle exposed to lengthening contractions compared with contralateral control muscle. Lengthening contractions also resulted in immediate translocation of constitutively expressed HSP25 and B-crystallin from the soluble to the insoluble fraction of muscle homogenates, and cryosections showed translocation from a diffuse, cytosolic localization to striations that corresponded to z-disks. Lengthening contraction-induced translocation of HSP25 and B-crystallin was associated with phosphorylation of these small HSPs, which may trigger their protective activity. In summary, these findings demonstrate loss of z-disk and membrane scaffolding proteins immediately after lengthening contractions, and concomitant translocation of HSP25 and B-crystallin to the z-disk, which may help to stabilize or repair cytoskeletal elements at this site. skeletal muscle injury; heat shock protein 25; B-crystallin; dystrophin; desmin     

Heterologous expression of Na(+)-K(+)-ATPase in insect cells: intracellular distribution of pump subunits

TheNa⁺-K⁺-ATPase is a heterodimeric plasmamembrane protein responsible for cellular ionic homeostasis in nearlyall animal cells. It has been shown that some insect cells (e.g., HighFive cells) have no (or extremely low)Na⁺-K⁺-ATPase activity. We expressed sheepkidney Na⁺-K⁺-ATPase - and -subunitsindividually and together in High Five cells via the baculovirusexpression system. We used quantitative slot-blot analyses to determinethat the expressed Na⁺-K⁺-ATPase comprisesbetween 0.5% and 2% of the total membrane protein in these cells.Using a five-step sucrose gradient (0.8-2.0 M) to separate theendoplasmic reticulum, Golgi apparatus, and plasma membrane fractions,we observed functional Na⁺ pump molecules in each membranepool and characterized their properties. Nearly all of the expressedprotein functions normally, similar to that found in purified dogkidney enzyme preparations. Consequently, the measurements describedhere were not complicated by an abundance of nonfunctionalheterologously expressed enzyme. Specifically, ouabain-sensitive ATPaseactivity, [³H]ouabain binding, and cation dependencieswere measured for each fraction. The functional properties of theNa⁺-K⁺-ATPase were essentially unaltered afterassembly in the endoplasmic reticulum. In addition, we measuredouabain-sensitive ⁸⁶Rb⁺ uptake in whole cellsas a means to specifically evaluateNa⁺-K⁺-ATPase molecules that were properlyfolded and delivered to the plasma membrane. We could not measure anyouabain-sensitive activities when either the -subunit or -subunitwere expressed individually. Immunostaining of the separate membranefractions indicates that the -subunit, when expressed alone, isdegraded early in the protein maturation pathway (i.e., the endoplasmicreticulum) but that the -subunit is processed normally and deliveredto the plasma membrane. Thus it appears that only the -subunit hasan oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the - heterodimer within theendoplasmic reticulum apparently does not require a Na⁺pump-specific chaperone.

     

Growth hormone regulates the distribution of L-type calcium channels in rat adipocyte membranes

Earlier studies demonstrated that deprivation of growth hormone(GH) for 3 h decreased basal and maximally stimulated cytosolic Ca²⁺ in rat adipocytes andsuggested that membrane Ca²⁺channels might be decreased. Measurement of L-typeCa²⁺ channels in purified plasmamembranes by immunoassay or dihydropyridine binding indicated a two- tofourfold decrease after 3 h of incubation without GH. No such decreasewas seen in unfractionated adipocyte membrane preparations. Thedecrease in plasma membrane channel content was largely accounted forby redistribution of channels to a light microsomal membrane fraction.Immunoassay of ₁-,₂/-, and -channelsubunits in membrane fractions indicated that the channelsredistributed as intact complexes. Addition of GH during the 1st h ofincubation prevented channel redistribution, and addition of GH after 3 h restored channel distribution to the GH-replete state of freshlyisolated adipocytes. The studies suggest that GH may regulate theabundance of Ca²⁺ channels in theadipocyte plasma membrane and thereby modulate sensitivity to signals,the expression of which is Ca²⁺dependent.

     

Characterization of PKA isoforms and kinase-dependent activation of chloride secretion in T84 cells

Chloride exitacross the apical membranes of secretory epithelial cells is acutelyregulated by the cAMP-mediated second messenger cascade. To betterunderstand the regulation of transepithelial chloride secretion, wehave characterized the complement of cAMP-dependent protein kinase(PKA) isoforms present in the human colonic epithelial cell line T84.Our results show that both type I and type II PKA are present in T84cells. Immunoprecipitation of8-azido-[³²P]cAMP-labeledcell lysates revealed that the major regulatory subunits of PKA wereRI and RII. In addition, immunogold electron microscopy showed that RII labeling was found on membranes of thetrans Golgi network and on apicalplasma membrane. In contrast, RI was randomly distributed throughoutthe cytoplasm, with no discernible membrane association. Northern blotanalysis of T84 RNA revealed that C was the predominantly expressedcatalytic subunit. Short-circuit current measurements were performed in the presence of combinations of site-selective cAMP analog pairs topreferentially activate either PKA type I or PKA type II in intact T84cell monolayers. Maximal levels of chloride secretion (~100µA/cm²) were observed for bothtype I and type II PKA-selective analog pairs. Subsequent addition offorskolin was unable to further increase chloride secretion. Thusactivation of either type I or type II PKA is able to maximallystimulate chloride secretion in T84 colonic epithelial cells.

     

The influence of Lyn kinase on Na,K-ATPase in porcine lens epithelium

Na,K-ATPase is essential for the regulation of cytoplasmic Na⁺ and K⁺ levels in lens cells. Studies on the intact lens suggest activation of tyrosine kinases may inhibit Na,K-ATPase function. Here, we tested the influence of Lyn kinase, a Src-family member, on tyrosine phosphorylation and Na,K-ATPase activity in membrane material isolated from porcine lens epithelium. Western blot studies indicated the expression of Lyn in lens cells. When membrane material was incubated in ATP-containing solution containing partially purified Lyn kinase, Na,K-ATPase activity was reduced by 38%. Lyn caused tyrosine phosphorylation of multiple protein bands. Immunoprecipitation and Western blot analysis showed Lyn treatment causes an increase in density of a 100-kDa phosphotyrosine band immunopositive for Na,K-ATPase ₁ polypeptide. Incubation with protein tyrosine phosphatase 1B (PTP-1B) reversed the Lyn-dependent tyrosine phosphorylation increase and the change of Na,K-ATPase activity. The results suggest that Lyn kinase treatment of a lens epithelium membrane preparation is able to bring about partial inhibition of Na,K-ATPase activity associated with tyrosine phosphorylation of multiple membrane proteins, including the Na,K-ATPase ₁ catalytic subunit. lens; Na,K-ATPase; tyrosine phosphorylation; Lyn     

Nicotinic acetylcholine receptor alpha 7 regulates cAMP signal within lipid rafts

Neuronal nicotinic acetylcholine receptors (nAChRs) are made of multiple subunits with diversified functions. The nAChR ₇-subunit has a property of high Ca²⁺ permeability and may have specific functions and localization within the plasma membrane as a signal transduction molecule. In PC-12 cells, fractionation by sucrose gradient centrifugation revealed that nAChR₇ existed in low-density, cholesterol-enriched plasma membrane microdomains known as lipid rafts where flotillin also exists. In contrast, nAChR ₅- and ₂-subunits were located in high-density fractions, out of the lipid rafts. Type 6 adenylyl cyclase (AC6), a calcium-inhibitable isoform, was also found in lipid rafts and was coimmunoprecipitated with nAChR₇. Cholesterol depletion from plasma membranes with methyl--cyclodextrin redistributed nAChR₇ and AC6 diffusely within plasma membranes. Nicotine stimulation reduced forskolin-stimulated AC activity by 35%, and this inhibition was negated by either treatment with -bungarotoxin, a specific antagonist of nAChR₇, or cholesterol depletion from plasma membranes. The effect of cholesterol depletion was negated by the addition of cholesterol. These data suggest that nAChR₇ has a specific membrane localization relative to other nAChR subunits and that lipid rafts are necessary to localize nAChR₇ with AC within plasma membranes. In addition, nAChR₇ may regulate the AC activity via Ca²⁺ within lipid rafts. cholesterol; PC-12 cells     

{alpha}Melanocyte Stimulating Hormone: Chemical Nature and Mechanism of Action

Melanotropin (-MSH) is a tridecapeptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂,synthesized and secreted by the pars intermedia of the vertebratepituitary. This peptide hormone is derived from pro-opiomelanocortin,a precursor protein which contains within its structure thesequences of other melanotropic peptides (- and rß-MSH,corticotropin), and possibly other hormones. -MSH is the physiologicallyrelevant melanotropin secreted by the pituitary and in mostvertebrates plays the essential role in adaptive color changesthrough its action on integumental chromatophores. The initial actions of -MSH are mediated at the level of themelanocyte membrane and involve signal transduction from receptorto adenylate cyclase on the intracellular surface of the membrane.This results in elevated cytosolic cyclic AMP levels followedby melanosome dispersion within dermal melanocytes and melanogenesiswithin epidermal melanocytes. The action of -MSH on dermal melanocytesrequires calcium for transduction of signal and cyclic AMP production.Melanosome dispersion per se does not, however, require extracellularcalcium. Structure-function studies of -MSH analogues and fragmentshave provided important insights relative to the structuralrequirements of the hormone for receptor binding and transduction.Substitution of certain residues within -MSH has led to thedevelopment of melanotropins that exhibit extraordinary potencyand prolonged biological activity     

Effects of Metal Chelators on Leaf Senescence in Maize and Hydrangea

The senescence of maize and hydrangea leaves after being detachedand kept in the dark was studied in terms of the loss of chlorophyll.Chlorophyll was more rapidly degraded in maize than hydrangeaduring the incubation period in the dark. The loss of chlorophyllin the dark was effectively inhibited in both plants by ,'-dipyridyland o-phenanthroline at concentrations between 0.01 and 0.1mM. Three other chelators of iron produced lesser inhibitionand only at higher concentrations. EDTA prevented the loss ofchlorophyll in maize leaves at concentrations above 10 mM, butdid not do so in hydrangea leaves. Detached leaves floated on EDTA, ,'-dipyridyl or o-phenanthrolinesolutions and exposed to light exhibited a marked bleaching.The bleaching was partially inhibited by applying ascorbic acid. (Received December 26, 1981; Accepted October 18, 1982)     

Transmembrane-Domain Trapping: A Novel Method for Isolation of cDNAs Encoding Putative Membrane Proteins

We have developed a method that enables us to isolate cDNAsof putative membrane proteins. The system is designed to isolatea cDNA which can provide the transmembrane domain to the extracellularpart of the IL-2 receptor chain. We constructed a p18Mac vectorby putting part of the IL-2 receptor chain cDNA that encodedits signal sequence and extracellular domain, a cDNA cloningsite and a poly(A) additional signal after a strong promoterSR. If a cloned cDNA provides a transmembrane domain in-frame,the extracellular domain of the IL-2 receptor chain will beexpressed on the surface of the transfected cells. Otherwise,the chimeric protein will be either secreted or retained insidethe transfected cells. We made a cDNA library using p18Mac andscreened for cDNA clones which allowed the expression of theextracellular domain of the IL-2 receptor chain on the cellsurface. Of the 2000 clones screened, 5 clones were scored aspositive. Partial sequence analysis revealed that one cloneencoded the amyloid precursor protein, two others encoded mitochondrialproteins and the rest were new. These results suggest the systemis effective in isolating cDNAs encoding putative membrane proteins.     

Hearing and glycoconjugates: localization of Ley, Lex and sialosyl-Lex in guinea pig cochlea, particularly at the tectorial membrane and sensory epithelia of the organ of Corti

Immunohistological examination of guinea pig cochleas was performedusing a panel of 25 monoclonal antibodies directed to variouslacto-, ganglio- and globo-series carbohydrate epitopes as wellas mucin-type epitopes. Lacto-series structures were found tobe localized at specific sites of the tectorial membrane (TM)and Corti's organ, i.e. 13 fucosyl type 2 chain (Le^x) at Kimura'smembrane, marginal band and covering net of TM; 12, 13 difucosyltype 2 chain (Le^y) at covering net; and sialosyl-Le^x and sialosyl-iat Kimura's membrane and sensory epithelia, particularly sensorytips of hair cells of Corti's organ. In striking contrast, ganglio-seriesstructures (GM3, GD3, GD2, 9-O-Ac-GD3) were detected at spiralganglion cells, neuronal fibres and stria vascularis, but werecompletely absent from Corti's organ and most of the TM. Otherepitope structures defined by various antibodies were not detectableat any location. The functional roles of lacto-series carbohydrateepitopes expressed at TM and Corti's organ remain unknown. However,the expression of Le^y (but not other structures) in associationwith developmental deficiency of TM induced by 6-N-propyI-2-thio-uracilin rats suggests that Le^y plays some role in normal TM development.The presence of Le^x at Kimura's membrane and sialosyl-Le^x athair cell sensory tips of Corti's organ suggests the intriguingpossibility that these fucosylated/sialosylated carbohydratestructures play some role in interactions (either attractiveor repulsive) of these inner ear components, which have beenimplicated in the physiology of hearing, i.e. the conversionof sound waves to nerve impulses. cochlea Corti's organ glycoconjugate sialosyl fucosyl type 2 chain tectorial membrane     

Differential translocation of protein kinase C isozymes by phorbol esters, EGF, and ANG II in rat liver WB cells

The protein kinaseC (PKC) family represents an important group of enzymes whoseactivation is associated with their translocation from the cytosol todifferent cellular membranes. In this study, the spatial distributionof PKC-, - and - in rat liver epithelial (WB) cells has beenexamined by Western blot analysis after subcellular fractionation.Cytosolic, membrane, nuclear, and cytoskeletal fractions were obtainedfrom cells stimulated with phorbol 12-myristate 13-acetate (PMA),angiotensin II (ANG II), or epidermal growth factor (EGF). PMA causedmost of the PKC-, - and - initially present in the cytosol tobe transported to the membrane and nuclear fractions. In contrast, bothANG II and EGF induced only a minor translocation of PKC- to themembrane fraction but caused a statistically significantmembrane-directed movement of PKC- and -. Translocation ofPKC- and - to the nucleus induced by ANG II and EGF was transient and quantitatively smaller than that induced by PMA. PKC- and -were present in the cytoskeleton of resting cells, but although PMA,ANG II, and EGF caused some changes in their content, these werevariable, suggesting that the cytoskeleton fraction was heterogeneous. PKC depletion inhibited ANG II-induced mitogenesis and the sustained activation of Raf-1 and extracellular regulated protein kinase (ERK).However, although PKC depletion inhibited EGF-induced mitogenesis, themaximum EGF-induced activation of the ERK pathway was only slightlyretarded. We hypothesize that PKC- and - are involved inmitogenesis via both ERK-dependent and ERK-independent mechanisms. These results support the notion that specific PKC isozymes exert spatially defined effects by virtue of their directed translocation todistinct intracellular sites.

     

HTP: a neural network-based method for predicting the topology of helical transmembrane domains in proteins

In this paper we describe a microcomputer program (HTP) forpredicting the location and orientation of -helical transmemhranesegments in integral membrane proteins. HTP is a neural network-basedtool which gives as output the protein membrane topology basedon the statistical propensity of residues to be located in externaland internal loops. This method, which uses single protein sequencesas input to the network system, correctly predicts the topologyof 71 out of 92 membrane proteins of putative membrane orientation,independently of the protein source.     

TNF-alpha potentiates the ion secretion induced by muscarinic receptor activation in HT29cl.19A cells

Chronic gastrointestinaldiseases such as ulcerative colitis and Crohn's disease arecharacterized by severe diarrhea. Mucosal biopsies of these patientsshow enhanced levels of cytokines, secreted by infiltrated inflammatorycells. In this study, we investigated the effect of the cytokine tumornecrosis factor- (TNF-) on ion secretion in human intestinalepithelial cells. The conventional microelectrode technique in the cellline HT29cl.19A was used, which allows for simultaneous measurements oftransepithelial potential difference and intracellular potentialdifference across the apical membrane. Preincubation (2-78 h) with10 ng/ml TNF- did not change basal secretory activity. However, thesecretory response to the muscarinic receptor agonist carbachol wasstrongly increased after exposure to TNF-. Application of theprotein kinase C (PKC) inhibitor GF 109203X (bisindolylmaleimide I)inhibited the response to carbachol as well as the TNF--potentiatedresponse, indicating that PKC mediates the effect of carbachol in thiscell line. Propranolol, a substance that inhibits the phospholipase D(PLD) pathway, strongly reduced the response to muscarinic stimulation and its potentiation by TNF-. The results indicate that activation of PLD is involved in ion secretion induced by muscarinic receptor activation and that TNF- can potentiate this pathway.

     

Translated Alu sequence determines nuclear localization of a novel catalytic subunit of casein kinase 2

Casein kinase 2 (CK2) is atetrameric enzyme constitutively expressed in all eukaryotic tissues.The two known isoforms of the catalytic subunit, CK2 and CK2',have been reported to have distinct tissue-dependent subcellulardistributions. We recently described a third isoform of the catalyticsubunit, designated CK2", which is highly expressed in liver.Immunoblot analysis of HuH-7 human hepatoma cell fractions as well asimmunofluorescent microscopy revealed that CK2" was exclusivelylocalized to the nucleus and preferentially associated with the nuclearmatrix. CK2 and CK2' were found in nuclear, membrane, andcytosolic compartments. Deletion of the carboxy-terminal 32 amino acids from the CK2" sequence resulted in release of the truncated green fluorescent protein fusion protein from the nuclear matrix and redistribution to both the nucleus and the cytoplasm. Demonstration that the carboxy terminus is necessary but not sufficient for nuclearretention indicates that the underlying mechanism of CK2" nuclearlocalization is dependent on the secondary structure of the holoenzymedirected by the carboxy-terminal sequence.

     

Adult alveolar epithelial cells express multiple subtypes of voltage-gated K+ channels that are located in apical membrane

Whole cell perforated patch-clampexperiments were performed with adult rat alveolar epithelial cells.The holding potential was 60 mV, and depolarizing voltage stepsactivated voltage-gated K⁺ (Kv) channels. Thevoltage-activated currents exhibited a mean reversal potential of 32mV. Complete activation was achieved at 10 mV. The currents exhibitedslow inactivation, with significant variability in the time coursebetween cells. Tail current analysis revealed cell-to-cell variabilityin K⁺ selectivity, suggesting contributions of multiple Kv-subunits to the whole cell current. The Kv channels also displayedsteady-state inactivation when the membrane potential was held atdepolarized voltages with a window current between 30 and 5 mV.Analysis of RNA isolated from these cells by RT-PCR revealed thepresence of eight Kv -subunits (Kv1.1, Kv1.3, Kv1.4, Kv2.2, Kv4.1,Kv4.2, Kv4.3, and Kv9.3), three -subunits (Kv1.1, Kv2.1, andKv3.1), and two K⁺ channel interacting protein (KChIP)isoforms (KChIP2 and KChIP3). Western blot analysis with available Kv-subunit antibodies (Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3) showedlabeling of 50-kDa proteins from alveolar epithelial cells grown inmonolayer culture. Immunocytochemical analysis of cells from monolayersshowed that Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3 were localized to theapical membrane. We conclude that expression of multiple Kv -, -,and KChIP subunits explains the variability in inactivation gating andK⁺ selectivity observed between cells and that Kv channelsin the apical membrane may contribute to basal K⁺ secretionacross the alveolar epithelium.

     

Ca2+-dependent inhibition of NHE3 requires PKC alpha which binds to E3KARP to decrease surface NHE3 containing plasma membrane complexes

The intestinal brush border (BB) Na⁺/H⁺ exchanger isoform 3 (NHE3) is acutely inhibited by elevation in the concentration of free intracellular Ca²⁺ ([Ca²⁺]_i) by the cholinergic agonist carbachol and Ca²⁺ ionophores in a protein kinase C (PKC)-dependent manner. We previously showed that elevating [Ca²⁺]_i with ionomycin rapidly inhibited NHE3 activity and decreased the amount of NHE3 on the plasma membrane in a manner that depended on the presence of the PDZ domain-containing protein E3KARP (NHE3 kinase A regulatory protein, also called NHERF2). The current studies were performed in PS120 fibroblasts (NHE-null cell line) stably transfected with NHE3 and E3KARP to probe the mechanism of PKC involvement in Ca²⁺ regulation of NHE3. Pretreatment with the general PKC inhibitor, GF109203X prevented ionomycin inhibition of NHE3 without altering basal NHE3 activity. Similarly, the Ca²⁺-mediated inhibition of NHE3 activity was blocked after pretreatment with the conventional PKC inhibitor Gö-6976 and a specific PKC pseudosubstrate-derived inhibitor peptide. [Ca²⁺]_i elevation caused translocation of PKC from cytosol to membrane. PKC bound to the PDZ1 domain of GST-E3KARP in vitro in a Ca²⁺-dependent manner. PKC and E3KARP coimmunoprecipitated from cell lysates; this occurred to a lesser extent at basal [Ca²⁺]_i and was increased with ionomycin exposure. Biotinylation studies demonstrated that [Ca²⁺]_i elevation induced oligomerization of NHE3 in total lysates and decreased the amount of plasma membrane NHE3. Treatment with PKC inhibitors did not affect the oligomerization of NHE3 but did prevent the decrease in surface amount of NHE3. These results suggest that PKC is not necessary for the Ca²⁺-dependent formation of the NHE3 plasma membrane complex, although it is necessary for decreasing the membrane amounts of NHE3, probably by stimulating NHE3 endocytosis. Na absorption; PDZ domains; signal complex     

GAIP, a Galpha i-3-binding protein, is associated with Golgi-derived vesicles and protein trafficking

Proteins of theregulators of G protein signaling (RGS) family bind to G subunits todownregulate their signaling in a variety of systems. G-interactingprotein (GAIP) is a mammalian RGS protein that shows high affinity forthe activated state of G_i-3, aprotein known to regulate post-Golgi trafficking of secreted proteins in kidney epithelial cells. This study aimed to localize GAIP inepithelial cells and to investigate its potential role in the regulation of membrane trafficking.LLC-PK₁ cells were stably transfected with a c-myc-tagged GAIPcDNA. In the transfected and untransfected cells, GAIP was found in thecytosol and on cell membranes. Immunogold labeling showed thatmembrane-bound GAIP was localized on budding vesicles around Golgistacks. When an in vitro assay was used to generate vesicles fromisolated rat liver and Madin-Darby canine kidney cell Golgi membranes, GAIP was found to be concentrated in fractions of newly budded Golgivesicles. Finally, the constitutive trafficking and secretion ofsulfated proteoglycans was measured in cell lines overexpressing GAIP.We show evidence for GAIP regulation of secretory trafficking beforethe level of the trans-Golgi networkbut not in post-Golgi secretion. The location and functional effects ofGAIP overlap only partially with those ofG_i-3 and suggest multiple roles for GAIP in epithelial cells.     

Cholinergic receptor-mediated differential cytoskeletal recruitment of actin- and integrin-binding proteins in intact airway smooth muscle

We tested the hypothesis that cholinergic receptor stimulation recruits actin- and integrin-binding proteins from the cytoplasm to the cytoskeleton-membrane complex in intact airway smooth muscle. We stimulated bovine tracheal smooth muscle with carbachol and fractionated the tissue homogenate into pellet (P) and supernatant (S) by ultracentrifugation. In unstimulated tissues, calponin exhibited the highest basal P-to-S ratio (P/S; 2.74 ± 0.47), whereas vinculin exhibited the lowest P/S (0.52 ± 0.09). Cholinergic receptor stimulation increased P/S of the following proteins in descending order of sensitivity: -actinin > talin metavinculin > -smooth muscle actin > vinculin calponin. Carbachol induced ERK1/2 phosphorylation by 300% of basal value. U0126 (10 µM) completely inhibited carbachol-induced ERK1/2 phosphorylation but did not significantly affect the correlation between -actinin P/S and carbachol concentration. This observation indicates that cytoskeletal/membrane recruitment of -actinin is independent of ERK1/2 mitogen-activated protein kinase activation. Metavinculin and vinculin are splice variants of a single gene, but metavinculin P/S was significantly higher than vinculin P/S. Furthermore, the P/S of metavinculin but not vinculin increased significantly in response to cholinergic receptor stimulation. Calponin and -actinin both belong to the family of calponin homology (CH) domain proteins. However, unlike -actinin, the calponin P/S did not change significantly in response to cholinergic receptor stimulation. These findings indicate differential cytoskeletal/membrane recruitment of actin- and integrin-binding proteins in response to cholinergic receptor stimulation in intact airway smooth muscle. -Actinin, talin, and metavinculin appear to be key cytoskeletal proteins involved in the recruitment process. actinin; mitogen-activated protein kinase; metavinculin; vinculin