A. Vaandrager | Utrecht University (original) (raw)

Papers by A. Vaandrager

Journal of Biological Chemistry, 1993

Guanylyl cyclase C (GC-C) is a newly discovered receptor found in the intestine, and possibly in ... more Guanylyl cyclase C (GC-C) is a newly discovered receptor found in the intestine, and possibly in other epithelia, that binds bacterial heat-stable enterotoxins (STa). The receptor has now been stably expressed in human embryonic 293 cells, which do not normally contain the receptor. Cyclic GMP concentrations are elevated 40-fold in response to 1 PM STa, and membranes obtained from the overproducing cells contain GC-C activity that can be stimulated about 9-fold by STa alone and an additional 1.4-to 2-fold by a combination of ATP and STa. The ATP effect does not appear to be due to enzyme activation, but instead to protection of GC-C against inactivation. Antibody raised against the carboxyl-terminal sequence of GC-C identified two major proteins (M, 140,000 and 160,000) in 293 cells expressing GC-C. Both proteins bound to wheat germ lectin-Sepharose, and N-glycosidase F treatment converted both forms to a single M, 120,000 protein, the size predicted from amino acid composition. The addition of high concentrations of tunicamycin to 293-GC-C cells also reduced the M, to 120,000, indicating that GC-C is an N-linked glycoprotein. When rat intestinal membranes or 293-GC-C cells were cross-linked with '261-labeled STa, the major '2SI-labeled protein complexes had M, ranging between 45,000 and 80,000. On immunoblots of rat intestinal membranes treated with a reducing agent, 3 major proteins of M, 65,000, 85,000, and 140,000 were specifically recognized by a GC-C antibody. However, under nonreducing conditions one major GC-C related protein appeared at a higher position (M, 230,000). Its mobility was reduced in the presence of STa, similar to rGC-C expressed in 293 cells. These data indicate that at least part of the lower M, STa-binding proteins found in intestinal extracts represent proteolytic products of GC-C. Secretory diarrhea caused by pathogenic strains of Escherichia coli is a major cause of infant deaths in developing countries (1). The enterotoxins secreted by these bacteria

American Journal of Physiology-Gastrointestinal and Liver Physiology, 1990

The potential role of polyphosphoinositide (PPI) metabolism as a signal-transduction mechanism in... more The potential role of polyphosphoinositide (PPI) metabolism as a signal-transduction mechanism in apical membranes of polarized epithelial cells was evaluated by examining the formation and breakdown of PPI in rat intestinal brush-border membranes (BBM) prelabeled by intraluminal injection of [3H]inositol in vivo or by [gamma-32P]ATP in vitro. Freshly isolated BBM prelabeled with [3H]inositol contained higher amounts of [3H]phosphatidylinositol 4,5-diphosphate compared with a basolateral membrane (BLM) preparation (approximately 14 and 6.8% of total [3H]PPIs, respectively) and were enriched in inositol lipid kinases, diacylglycerol (DAG) kinase, and phosphomonoesterases degrading PPI, inositol bisphosphate, and inositol triphosphate (IP3). In the presence of nonhydrolysable GTP analogues and low Ca2+ (pCa 6-8) or at high Ca2+ alone (pCa 4) endogenous pools of PPI were rapidly depleted by an intrinsic PPI-specific phospholipase C apparently coupled to a GTP-binding protein (G protein...

American Journal of Physiology-Gastrointestinal and Liver Physiology, 1991

The mechanism of adenosine 3',5'-cyclic monophosphate (cAMP)- and Ca(2+)-induced Cl- secr... more The mechanism of adenosine 3',5'-cyclic monophosphate (cAMP)- and Ca(2+)-induced Cl- secretion was studied in monolayers of the colon carcinoma cell line HT-29.cl19A by combined short-circuit current (Isc) and 125I- or 36Cl- efflux measurements. Forskolin, a specific adenylate cyclase activator, was found to induce a large increase in Isc and a two- to threefold increase in 36Cl- efflux solely across the apical border. The fractional efflux of 36Cl-compared with 125I- (basal ratio 1.71 +/- 0.28) did not change significantly in the presence of forskolin (1.91 +/- 0.45). In contrast, the Ca2+ ionophore A23187 did not appreciably affect the Isc but enhanced 36Cl- and 125I- efflux at the apical and basolateral side of the monolayer. Furthermore, the fractional efflux ratio of 36Cl- to 125I- changed dramatically to a value of 0.36 +/- 0.14. Both forskolin- and A23187-induced 36Cl- or 125I- efflux were only weakly inhibited by the putative Cl- channel blocker 5-nitro-2-(3-phenylpr...

American Journal of Physiology-Gastrointestinal and Liver Physiology, 1992

Phorbol 12-myristate 13-acetate (PMA) was found to increase both the short-circuit current (Isc) ... more Phorbol 12-myristate 13-acetate (PMA) was found to increase both the short-circuit current (Isc) and the efflux of 125I- or 36Cl- in the colonic epithelial cell line HT-29.cl19A. Neither the PMA-provoked rise in Isc nor the stimulation of 125I- efflux was affected by the cyclooxygenase inhibitor indomethacin. The PMA-induced increase in Cl- efflux was not accompanied by a rise in adenosine 3',5'-cyclic monophosphate (cAMP) levels. A prolonged incubation with PMA (3 h), however, inhibited the PMA- and the cAMP-stimulated Isc by greater than 90%, whereas the cAMP-provoked 125I- and 36Cl- efflux was not inhibited. The long-term PMA treatment was found to inhibit the basal and cAMP-provoked 86Rb+ efflux by 65 +/- 9 and 86 +/- 7%, respectively. A 3-h incubation with PMA also strongly inhibited the Ca2+ ionophore A23187-induced increase in 86Rb+ efflux, whereas the A23187-stimulated 125I- efflux was only marginally inhibited. These data suggest that phorbol esters, presumably by a...

The FASEB Journal, 1999

Nitric oxide (NO) and cGMP have been implicated in many neuronal functions, including regulation ... more Nitric oxide (NO) and cGMP have been implicated in many neuronal functions, including regulation of gene expression, but little is known about the downstream targets of NO/cGMP in the nervous system. We found that type II cGMP-dependent protein kinase (G-kinase), which is widely expressed in the brain, mediated NO-and cGMPinduced activation of the fos promoter in cells of neuronal and glial origin; the enzyme was ineffective in regulating gene expression in fibroblast-like cells. The effect of G-kinase II on gene expression did not require calcium uptake but was synergistically enhanced by calcium. G-kinase II was membrane associated and did not translocate to the nucleus; however, a soluble G-kinase II mutant translocated to the nucleus and regulated gene expression in fibroblastlike cells. Soluble G-kinase I also regulates fos promoter activity, but membrane targeting of G-kinase I prevented the enzyme from translocating to the nucleus and regulating transcription in multiple cell types, including glioma cells; this suggests that cell type-specific factor(s) that mediate the transcriptional effects of extranuclear G-kinase II are not regulated by G-kinase I. Our results suggest that G-kinase I and II control gene expression by different mechanisms and that NO effects on neuronal plasticity may involve G-kinase II regulation of gene expression.

Biochemical Journal, 1992

The involvement of protein kinase C (PKC) in the regulation of intestinal ion secretion was studi... more The involvement of protein kinase C (PKC) in the regulation of intestinal ion secretion was studied in polarized monolayers of the HT29cl.19A human colon carcinoma cell line. Carbachol, phorbol esters [PMA (phorbol 12-myristate 13-acetate) and PDB (phorbol 12,13-dibutyrate)] and 8-bromo cyclic AMP (8-Br-cAMP) induced Cl secretion, as measured by a rise in the short-circuit current (ISC). The electrical response to carbachol coincided with a transient translocation of PKC alpha from the soluble to the particulate fraction. The carbachol-, PDB- and 8-Br-cAMP-induced ISC responses were inhibited by pretreatment of the cells with PMA (0.5 microM) for 2 h, a time period in which PKC alpha, beta 1 and gamma levels were not changed. As shown by 86Rb+ and 125I- efflux studies, the main targets for this inhibition were basolateral K+ transporters rather than apical Cl- channels. Prolonged exposure to PMA (24 h) led to a 60% recovery of the 8-Br-cAMP response, but not of the carbachol- or PDB...

Gastroenterology, 1992

Studies with Escherichia co&induced heat-stable enterotoxin (ST,), an activator of intestinal par... more Studies with Escherichia co&induced heat-stable enterotoxin (ST,), an activator of intestinal particulate guanylate cyclase, have established an independent role for cyclic guanosine monophosphate (cGMP) as an intracellular mediator of intestinal salt and water secretion. The present study addressed whether atriopeptins (APs), known activators of particulate guanylate cyclase in other tissues, function as physiological agonists for cGMP-linked Cl-secretion in intestine. APs, in contrast to ST,, caused no or only minor changes in cGMP levels in freshly isolated rat intestinal villus and crypt cells and in cultured human colon carcinoma cell lines (HT29glc-, CaCo-2, and T84). Conversely, APs, but not ST,, induced a large increase in intracellular cGMP levels in the undifferentiated small intestinal cell lines IEC-8, IEC-18, and INT407. Addition of AP II (atria1 natriuretic peptide fragment 5-27) to stripped mucosa of rat proximal colon in Ussing chambers caused a transient increase in the transepithelial potential difference (PD), which most likely represents an increase in Cl-secretion. In contrast, a sustained increase in PD was observed in response to ST, or IBr-cGMP. The AP II-provoked increase in PD was blocked by the neurotoxin tetrodotoxin. Immunohistochemical detection of cGMP in this tissue provided evidence for a different localization pattern of cells responding with an increase in cGMP levels to ST, (colonocytes and goblet cells) or AP (specific cells in the submucosa) in rat proximal colon. This indicates that APs, unlike ST,,, do not directly stimulate the colonic epithelial cells but possibly provoke Clsecretion by release of a neurotransmitter in the submucosa.

Biochemical Journal, 1991

The distribution of the alpha and beta subunits of guanosine-nucleotide-binding proteins (G-prote... more The distribution of the alpha and beta subunits of guanosine-nucleotide-binding proteins (G-proteins) among the apical and basolateral membranes of polarized rat enterocytes was investigated by ADP-ribosylation assays in vitro and immunoblotting with G-protein-subunit-specific antisera. The enterocytes were found to express alpha i2, alpha ji3, alpha s and beta subunits, whereas alpha i1 and alpha o subunits could not be detected. The alpha i2 and alpha i3 subunits were located predominantly in the basolateral membrane, in contrast with the alpha s and beta subunits, which were distributed uniformly among both membranes. Furthermore, 39 kDa and 78 kDa proteins, recognized by anti-alpha i1/2 but not anti-alpha i1 or anti-alpha i3 specific antisera, and resistant to ADP-ribosylation by pertussis toxin, were localized exclusively at the apical border. These Gi-related proteins might represent novel members of the G-protein family. Activation of apical G-proteins by GTP or its analogues...

Comparative Veterinary Pharmacology, Toxicology and Theraphy, 1986

The absorption of electrolytes and water by mammalian small intestine and proximal colon is prima... more The absorption of electrolytes and water by mammalian small intestine and proximal colon is primarily controlled by an e1ectroneutra1 entry mechanism for Na+ and Cl− in the brush-border membrane of the enterocytes, consisting of parallel Na+/H+ and Cl−/HCO3 − exchangers coupled by circular proton movements. Enterotoxins produced by non-invasive micro-organisms (such as choleratoxin, heat-stable and heat-labile Escherichia coli toxin) and neurohumoral factors (such as acetylcholine, vasoactive intestinal peptide[VIP], prostaglandins, bradykinin) promote salt and water secretion by a dual action involving (1) blockade of the antiporters in the villus compartment and (2) Cl− channel opening in the apical membrane of intestinal crypt cells, allowing CT exit down an electrochemical gradient created by a basolateral Na-K-Cl2 entry process. During Cl− secretion, the accumulation of Na+ and K+ in the enterocyte is prevented by Na+ exit through the Na+/K+-pump and K+ exit through secretagogue-sensitive K+ channels in the basolateral membrane.

Advances in Pharmacology, 1994

Publisher Summary Cyclic guanosine monophosphate (cGMP) was established as an important regulator... more Publisher Summary Cyclic guanosine monophosphate (cGMP) was established as an important regulator of intestinal ion transport in the late seventies, when it was shown to be the intracellular mediator of salt and water secretion induced by Escherichia coli heat-stable enterotoxin (STa). The recent finding of a distinctive endogenous activator of intestinal guanylyl cyclase (guanylin) affirms that the intestine possesses a unique cGMP-signaling pathway for the physiological regulation of ion transport. The regulation of fluid secretion by cGMP, however, has been thought to be restricted to the intestine. However, the recent detection of some of the components of the “intestinal cGMP pathway” (GC-C and guanylin) in other tissues suggests that it is expressed more generally than previously assumed. The chapter discusses the role of cGMP pathway in the intestine as a regulator of transepithelial ion and fluid transport.

The Veterinary Journal, 2014

The first aim of this study was to determine whether vitamin D supplementation influenced the eff... more The first aim of this study was to determine whether vitamin D supplementation influenced the effects of high vitamin A intake on new bone formation in adult cats. The second aim was to determine whether high vitamin A intake in cats caused liver pathology and, if so, whether the current upper limit for the dietary intake of vitamin A for healthy adult cats would be safe. Twenty-four healthy adult cats were divided into four groups that received a control diet supplemented with peanut oil (control), or peanut oil containing a 100-fold increase in vitamin A (HA), or a 100-fold increase in vitamin A and a fivefold increase in vitamin D (HAMD), or a 100-fold increase in vitamin A and a 65-fold increase in vitamin D (HAHD) over a period of 18 months. Cats did not show abnormal locomotion or clinical signs of liver failure after 18 months of supplementation but did show subtle skeletal changes and liver pathology, suggesting that the current National Research Council (2006) safe upper limit for vitamin A for cats is too high. The addition of vitamin D did not seem to influence bone pathology. While moderately elevated dietary vitamin D levels (HAMD) seemed to protect cats against the liver pathology caused by the consumption of large amounts of vitamin A, higher dietary levels of vitamin D (HAHD) did not seem to be protective.

Trends in Biochemical Sciences, 1997

I wre a 100 aa cGK I(; DG2, T3 (la) DG2, T2a cGK II DGl Amino acid sequences derived from cDNAs f... more I wre a 100 aa cGK I(; DG2, T3 (la) DG2, T2a cGK II DGl Amino acid sequences derived from cDNAs for CGFnP-dependent protein kinase (cGK) IS (human placenta) and cGK II (rat intestine) (reviewed in Refs 2-4) were compared with the sequence of bovine lung cGK laR as standard. The Drosophila sequences selected for comparison were those most similar to cGK I [DGZ T3 (la) and T2a] and cGK II (DGl), respectively. Percentage identities for complete sequences (left) and those for internal domains are shown. Internal domains shown are those for dimerization (Dimer.), cGMP binding (sites 1 and 2), ATP binding and catalytic activity, and an unspecified carboxy-terminal domain.

Proceedings of the National Academy of Sciences, 1998

A recently cloned isoform of cGMP-dependent protein kinase (cGK), designated type II, was implica... more A recently cloned isoform of cGMP-dependent protein kinase (cGK), designated type II, was implicated as the mediator of cGMP-provoked intestinal Cl − secretion based on its localization in the apical membrane of enterocytes and on its capacity to activate cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channels. In contrast, the soluble type I cGK was unable to activate CFTR in intact cells, although both cGK I and cGK II could phosphorylate CFTR in vitro . To investigate the molecular basis for the cGK II isotype specificity of CFTR channel gating, we expressed cGK II or cGK I mutants possessing different membrane binding properties by using adenoviral vectors in a CFTR-transfected intestinal cell line, and we examined the ability of cGMP to phosphorylate and activate the Cl − channel. Mutation of the cGK II N-terminal myristoylation site (Gly 2 → Ala) reduced cGK II membrane binding and severely impaired cGK II activation of CFTR. Conversely, a chimeric protein, in...

Proceedings of the National Academy of Sciences, 1999

Atrial natriuretic peptide (ANP) and nitric oxide (NO) are key regulators of ion and water transp... more Atrial natriuretic peptide (ANP) and nitric oxide (NO) are key regulators of ion and water transport in the kidney. Here, we report that these cGMP-elevating hormones stimulate Ca 2+ reabsorption via a novel mechanism specifically involving type II cGMP-dependent protein kinase (cGK II). ANP and the NO donor, sodium nitroprusside (SNP), markedly increased Ca 2+ uptake in freshly immunodissected rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). Although readily increasing cGMP, ANP and SNP did not affect Ca 2+ and Na + reabsorption in primary cultures of these segments. Immunoblot analysis demonstrated that cGK II, and not cGK I, was present in freshly isolated CNT and CCD but underwent a complete down-regulation during the primary cell culture. However, upon adenoviral reexpression of cGK II in primary cultures, ANP, SNP, and 8-Br-cGMP readily increased Ca 2+ reabsorption. In contrast, no cGMP-dependent effect on electrogenic Na + transport was observed. The membr...

Molecular and Cellular Biochemistry, 1996

The second messenger cGMP is a major intracellular mediator of the vaso-active agents nitric oxid... more The second messenger cGMP is a major intracellular mediator of the vaso-active agents nitric oxide and natriuretic peptides. The principal targets ofcGMP are (i) phosphodiesterases, resulting in interference with the cAMP-signalling pathway, (ii) cGMPgated cation channels, and (iii) cGMP-dependent protein kinases (cGKs). Only two mammalian isotypes of cGK have been described so far: type I cGK, consisting of an ct and a [3 isoform, presumably splice variants of a single gene, and identified as the most prominent cGK isotype in the cardio-vascular system; and type II cGK, expressed mainly in the intestine, the kidney and the brain. High levels of cGK I are found in vascular smooth muscle cells, endothelial cells and platelets. In these cells, cGK I is thought to counteract the increase in contraction provoked by Ca-mobilizing agonists, to reduce endothelial permeability and to inhibit platelet aggregation, respectively. Relatively low levels ofcGK I are found in cardiomyocytes. In this cell type, cGK is implicated in the negative inotropic effect of cGMP, presumably through modulation of Ca channels and by diminishing the Ca-sensitivity of contractile proteins.

The Journal of Membrane Biology, 1992

The modulation of ion transport pathways in filtergrown monolayers of the CI-secreting subclone (... more The modulation of ion transport pathways in filtergrown monolayers of the CI-secreting subclone (19A) of the human colon carcinoma cell line HT-29 by muscarinic stimulation was studied by combined Ussing chamber and microimpalement experiments. Basolateral addition of 10 4 M carbachol induced a complex poly-phasic change of the cell potential consisting of (i) a fast and short (30-sec) depolarization of 15 + 1 mV from a resting value of-52-+ l mV and an increase of the fractional resistance of the apical membrane (first phase), (ii) a repolarization of 22 + I mV leading to a hyperpolarization of the cell (second phase), (iii) a depolarization of l l-+ 1 mV and a decrease of the fractional resistance of the apical membrane (the third phase), (iv) and sometimes, a hyperpolarization of 6 +-1 mV and an increase of the fractional resistance of the apical membrane (fourth phase). The transepithelial potential increased with a peak value of 2.4 + 0.3 mV (basolateral side positive). The transepithelial PD started to increase (serosa positive), coinciding with the start of the second phase of the intracellular potential change, and continued to increase during the third phase, lon replacements and electrical circuit analyses indicate that the first phase is caused by increase of the CI conductance in the apical and basolateral membrane, the second phase by increased K-conductance of the basolateral membrane, and the third phase and the fourth phase by increase and decrease, respectively, of an apical CI-conductance. The first and second phase of the carbachol effect could be elicited also by ionomycin. They were strongly reduced by EGTA. Phorbol dibutyrate (PDB) induced a sustained depolarization of the cell and a decrease of the apical fractional resistance. The results suggest that two different types of CI-channels are involved in the carbachol response: one Ca 2 § dependent and a second which may be PKC sensitive. In the presence of a supramaximal concentration of forskolin, carbachol evoked a further increase of the apical CIconductance. It is concluded that the short-lasting carbachol/Ca 2 ~-dependent C1-conductance is different from the forskolin-activated conductance. The increase of the CI conductance in the presence of forskolin by carbachol may be due to activation of different CI channels or to modulation of the PKA-activated CI channels by activated PKC.

The Journal of Immunology, 2003

Influenza A virus (IAV) infections are a major cause of respiratory disease of humans and animals... more Influenza A virus (IAV) infections are a major cause of respiratory disease of humans and animals. Pigs can serve as important intermediate hosts for transmission of avian IAV strains to humans, and for the generation of reassortant strains; this may result in the appearance of new pandemic IAV strains in humans. We have studied the role of the porcine lung collectins surfactant proteins D and A (pSP-D and pSP-A), two important components of the innate immune response against IAV. Hemagglutination inhibition assays revealed that both pSP-D and pSP-A display substantially greater inhibitory activity against IAV strains isolated from human, swine, and horse, than lung collectins from other animal species. The more potent activity of pSP-D results from interactions mediated by the asparagine-linked oligosaccharide located in the carbohydrate recognition domain of pSP-D, which is absent in SP-Ds from other species characterized to date. Presence of this sialylated oligosaccharide moiety...

Journal of Clinical Investigation, 1995

Certain pathogenic bacteria produce a family of heat stable enterotoxins (STa) which activate int... more Certain pathogenic bacteria produce a family of heat stable enterotoxins (STa) which activate intestinal guanylyl cyclases, increase cGMP, and elicit life-threatening secretory diarrhea. The intracellular effector of cGMP actions has not been clarified. Recently we cloned the cDNA for a rat intestinal type II cGMP dependent protein kinase (cGK II) which is highly enriched in intestinal mucosa. Here we show that cGK II mRNA and protein are restricted to the intestinal segments from the duodenum to the proximal colon, with the highest amounts of cGK II protein in duodenum and jejunum. cGK II mRNA and protein decreased along the villus to crypt axis in the small intestine, whereas substantial amounts of both were found in the crypts of cecum. In intestinal epithelia, cGK II was specifically localized in the apical membrane, a major site of ion transport regulation. In contrast to cGK II, cGK I was localized in smooth muscle cells of the villus lamina propria. Short circuit current (ISc), a measure of Cl-secretion, was increased to a similar extent by STa and by 8-Br-cGMP, a selective activator of cGK, except in distal colon and in monolayers of T84 human colon carcinoma cells in which cGK II was not detected. In human and mouse intestine, the cyclic nucleotide-regulated Clconductance can be exclusively accounted for by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel. Viewed collectively, the data suggest that cGK II is the mediator of STa and cGMP effects on Cltransport in intestinal epithelia. (J.

Journal of Biological Chemistry, 1997

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-pr... more In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl ؊ secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl ؊ channels was analyzed after expression of cGK II or cGK I␤ in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125 I ؊ efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK I␤ or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK I␤ enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are colocalized, e.g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK I␤ nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.

Journal of Biological Chemistry, 1996

The apical membrane of intestinal epithelial cells harbors a unique isozyme of cGMP-dependent pro... more The apical membrane of intestinal epithelial cells harbors a unique isozyme of cGMP-dependent protein kinase (cGK type II) which acts as a key regulator of ion transport systems, including the cystic fibrosis transmembrane conductance regulator (CFTR)-chloride channel. To explore the mechanism of cGK II membrane-anchoring, recombinant cGK II was expressed stably in HEK 293 cells or transiently in COS-1 cells. In both cell lines, cGK II was found predominantly in the particulate fraction. Immunoprecipitation of solubilized cGK II did not reveal any other tightly associated proteins, suggesting a membrane binding motif within cGK II itself. The primary structure of cGK II is devoid of hydrophobic transmembrane domains; cGK II does, however, contain a penultimate glycine, a potential acceptor for a myristoyl moiety. Metabolic labeling showed that cGK II was indeed able to incorporate [ 3 H]myristate. Moreover, incubation of cGK II-expressing 293 cells with the myristoylation inhibitor 2-hydroxymyristic acid (1 mM) significantly increased the proportion of cGK II in the cytosol from 10 ؎ 5 to 35 ؎ 4%. Furthermore, a nonmyristoylated cGK II Gly 2 3 Ala mutant was localized predominantly in the cytosol after transient expression in COS-1 cells. The absence of the myristoyl group did not affect the specific enzyme activity or the K a for cGMP and only slightly enhanced the thermal stability of cGK II. These results indicate that N-terminal myristoylation fulfills a crucial role in directing cGK II to the membrane.