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PubMed2026

Mechanistic trade-offs between local and long-range signaling activity in natural and synthetic morphogens.

Hedgehog family morphogens present an interesting paradox: Despite being hydrophobic because of dual-lipid modifications, they form spatial concentration gradients that are highly conserved and essential for many aspects of metazoan development. Using live-cell single-molecule tracking and engineered synthetic signaling ligands, we isolated the distinct contribution of each lipid modification to Hedgehog diffusion and signaling potency. We found that although both lipid modifications enhance signaling potency, they do so through different mechanisms. Palmitate directly promotes receptor engagement, whereas cholesterol topologically confines secreted morphogens on the cell surface, effectively using the lipid membrane as a nonsignaling co-receptor that enriches ligands locally at the cost of restricting long-range diffusion. Our results on the function of cholesterol point to an intrinsic trade-off between signaling potency and gradient formation, with implications for the evolution and mechanism of nonsignaling co-receptors.

Signal Transduction↗
PubMed1999

Gbetagamma and palmitate target newly synthesized Galphaz to the plasma membrane.

The subcellular location of a signaling protein determines its ability to transmit messages accurately and efficiently. Three different lipid modifications tether heterotrimeric G proteins to membranes: alpha subunits are myristoylated and/or palmitoylated, and gamma subunits are prenylated. In a previous study, we examined the role of lipid modifications in maintaining the membrane attachment of a G protein alpha subunit, alphaz, which is myristoylated and palmitoylated (Morales, J., Fishburn, C. S., Wilson, P. T., and Bourne, H. R. (1998) Mol. Biol. Cell 9, 1-14). Now we extend this analysis by characterizing the mechanisms that target newly synthesized alphaz to the plasma membrane (PM) and analyze the role of lipid modifications in this process. In comparison with newly synthesized alphas, which is palmitoylated but not myristoylated, alphaz moves more rapidly to the membrane fraction following synthesis in the cytosol. Newly synthesized alphaz associates randomly with cellular membranes, but with time accumulates at the PM. Palmitoylated alphaz is present only in PM-enriched fractions, whereas a nonpalmitoylated mutant of alphaz (alphazC3A) associates less stably with the PM than does wild-type alphaz. Expression of a C-terminal fragment of the beta-adrenoreceptor kinase, which sequesters free betagamma, impairs association of both alphaz and alphazC3A with the PM, suggesting that the alpha subunit must bind betagamma in order to localize at the PM. Based on these findings, we propose a model in which, following synthesis on soluble ribosomes, myristoylated alphaz associates randomly and reversibly with membranes; upon association with the PM, alphaz binds betagamma, which promotes its palmitoylation, thus securing it in the proper place for transmitting the hormonal signal.

Animals↗
PubMed2005

Crystal structure and mechanism of the Escherichia coli ArnA (PmrI) transformylase domain. An enzyme for lipid A modification with 4-amino-4-deoxy-L-arabinose and polymyxin resistance.

Gram-negative bacteria have evolved mechanisms to resist the bactericidal action of cationic antimicrobial peptides of the innate immune system and antibiotics such as polymyxin. The strategy involves the addition of the positively charged sugar 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A in their outer membrane. ArnA is a key enzyme in the Ara4N-lipid A modification pathway. It is a bifunctional enzyme catalyzing (1) the oxidative decarboxylation of UDP-glucuronic acid (UDP-GlcA) to the UDP-4' '-ketopentose [UDP-beta-(l-threo-pentapyranosyl-4' '-ulose] and (2) the N-10-formyltetrahydrofolate-dependent formylation of UDP-Ara4N. Here we demonstrate that the transformylase activity of the Escherichia coli ArnA is contained in its 300 N-terminal residues. We designate it the ArnA transformylase domain and describe its crystal structure solved to 1.7 A resolution. The enzyme adopts a bilobal structure with an N-terminal Rossmann fold domain containing the N-10-formyltetrahydrofolate binding site and a C-terminal subdomain resembling an OB fold. Sequence and structure conservation around the active site of ArnA transformylase and other N-10-formyltetrahydrofolate-utilizing enzymes suggests that the HxSLLPxxxG motif can be used to identify enzymes that belong to this family. Binding of an N-10-formyltetrahydrofolate analogue was modeled into the structure of ArnA based on its similarity with glycinamide ribonucleotide formyltransferase. We also propose a mechanism for the transformylation reaction catalyzed by ArnA involving residues N(102), H(104), and D(140). Supporting this hypothesis, point mutation of any of these residues abolishes activity.

Amino Acid Sequence↗
PubMed2003

Prediction of lipid posttranslational modifications and localization signals from protein sequences: big-Pi, NMT and PTS1.

Many posttranslational modifications (N-myristoylation or glycosylphosphatidylinositol (GPI) lipid anchoring) and localization signals (the peroxisomal targeting signal PTS1) are encoded in short, partly compositionally biased regions at the N- or C-terminus of the protein sequence. These sequence signals are not well defined in terms of amino acid type preferences but they have significant interpositional correlations. Although the number of verified protein examples is small, the quantification of several physical conditions necessary for productive protein binding with the enzyme complexes executing the respective transformations can lead to predictors that recognize the signals from the amino acid sequence of queries alone. Taxon-specific prediction functions are required due to the divergent evolution of the active complexes. The big-Pi tool for the prediction of the C-terminal signal for GPI lipid anchor attachment is available for metazoan, protozoan and plant sequences. The myristoyl transferase (NMT) predictor recognizes glycine N-myristoylation sites (at the N-terminus and for fragments after processing) of higher eukaryotes (including their viruses) and fungi. The PTS1 signal predictor finds proteins with a C-terminus appropriate for peroxisomal import (for metazoa and fungi). Guidelines for application of the three WWW-based predictors (http://mendel.imp.univie.ac.at/) and for the interpretation of their output are described.

Acyltransferases↗
PubMed2005

Lipid posttranslational modifications. Farnesyl transferase inhibitors.

Some proteins undergo posttranslational modification by the addition of an isoprenyl lipid (farnesyl- or geranylgeranyl-isoprenoid) to a cysteine residue proximal to the C terminus. Protein isoprenylation promotes membrane association and contributes to protein-protein interactions. Farnesylated proteins include small GTPases, tyrosine phosphatases, nuclear lamina, cochaperones, and centromere-associated proteins. Prenylation is required for the transforming activity of Ras. Because of the high frequency of Ras mutations in cancer, farnesyl transferase inhibitors (FTIs) were investigated as a means to antagonize Ras function. Evaluation of FTIs led to the finding that both K- and N-Ras are alternatively modified by geranylgeranyl prenyltransferase-1 in FTI-treated cells. Geranylgeranylated forms of Ras retain the ability to associate with the plasma membrane and activate substrates. Despite this, FTIs are effective at inhibiting the growth of human tumor cells in vitro, suggesting that activity is dependent on blocking the farnesylation of other proteins. FTIs also inhibit the in vivo growth of human tumor xenografts and sensitize these models to chemotherapeutics, most notably taxanes. Several FTIs have entered clinical trials for various cancer indications. In some clinical settings, primarily hematologic malignancies, FTIs have displayed evidence of single-agent activity. Clinical studies in progress are exploring the antitumor activity of FTIs as single agents and in combination. This review will summarize the basic biology of FTIs, their antitumor activity in preclinical models, and the current status of clinical studies with these agents.

Animals↗
PubMed2000

Activation of H-ras61L-specific signaling pathways does not require posttranslational processing of H-ras.

We have previously demonstrated that H-ras61L retained transforming activity when lacking C-terminal lipid modifications, provided that plasma membrane localization was restored by an N-terminal transmembrane domain. Since several ras-activated pathways contribute to the transformed phenotype, we utilized a novel set of transmembrane domain-anchored H-ras derivatives to examine if lipids are required for activation of any specific signaling pathways. We demonstrate here that H-ras61L-induced activation of the Raf/MEK/MAPK pathway, including recruitment of Raf to the plasma membrane and activation of Raf and MAPK, does not require C-terminal processing of H-ras61L. Biochemical fractionation experiments confirm the localization of TM-ras derivatives to the plasma membrane, as well as the ras-mediated recruitment of c-Raf-1. Changes in the actin cytoskeleton, controlled by H-ras61L-mediated activation of the Rac/ Rho pathway, as well as PI 3-kinase activation, can also occur in the absence of C-terminal lipid modifications. Finally, downstream events, such as the induction of the immediate-early gene c-fos or neurite outgrowth in PC12 cells, are stimulated by the expression of plasma membrane-anchored, nonlipidated H-ras6lL. These results demonstrate that H-ras can be functionally targeted to the plasma membrane using a transmembrane domain sequence and that several signal transduction pathways downstream of H-ras can be activated without the presence of normal lipid modifications.

Animals↗
PubMed2006

Thematic review series: lipid posttranslational modifications. Structural biology of protein farnesyltransferase and geranylgeranyltransferase type I.

More than 100 proteins necessary for eukaryotic cell growth, differentiation, and morphology require posttranslational modification by the covalent attachment of an isoprenoid lipid (prenylation). Prenylated proteins include members of the Ras, Rab, and Rho families, lamins, CENPE and CENPF, and the gamma subunit of many small heterotrimeric G proteins. This modification is catalyzed by the protein prenyltransferases: protein farnesyltransferase (FTase), protein geranylgeranyltransferase type I (GGTase-I), and GGTase-II (or RabGGTase). In this review, we examine the structural biology of FTase and GGTase-I (the CaaX prenyltransferases) to establish a framework for understanding the molecular basis of substrate specificity and mechanism. These enzymes have been identified in a number of species, including mammals, fungi, plants, and protists. Prenyltransferase structures include complexes that represent the major steps along the reaction path, as well as a number of complexes with clinically relevant inhibitors. Such complexes may assist in the design of inhibitors that could lead to treatments for cancer, viral infection, and a number of deadly parasitic diseases.

Alkyl and Aryl Transferases↗
PubMed2000

Function of the membrane fusion protein, MexA, of the MexA, B-OprM efflux pump in Pseudomonas aeruginosa without an anchoring membrane.

Resistance of Pseudomonas aeruginosa to multiple species of antibiotics is largely attributable to expression of the MexA, B-OprM efflux pump. The MexA protein is thought to be located at the inner membrane and has been assumed to link the xenobiotics-exporting subunit, MexB, and the outer membrane channel protein, OprM. To verify this assumption, we analyzed membrane anchoring and localization of the MexA protein. n-[9, 10-(3)H]Palmitic acid incorporation experiments revealed that MexA was radiolabeled with palmitic acid, suggesting that the MexA anchors the inner membrane via the fatty acid moiety. To evaluate the role of lipid modification and inner membrane anchoring, we substituted cysteine 24 with phenylalanine or tyrosine and tested whether or not these mutant MexAs function properly. When the mutant mexAs were expressed in the strain lacking chromosomal mexA in the presence of n-[9,10-(3)H]palmitic acid, we found undetectable radiolabeling at the MexA band. These transformants restored antibiotic resistance to the level of the wild-type strain, indicating that lipid modification is not essential for MexA function. These mutant strains contained both processed and unprocessed forms of the MexA proteins. Cellular fractionation experiments revealed that an unprocessed form of MexA anchored the inner membrane probably via an uncleaved signal sequence, whereas the processed form was undetectable in the membrane fraction. To assure that the lipid-free MexA polypeptide could be unbound to the membrane, we analyzed the two-dimensional membrane topology by the gene fusion technique. A total of 78 mexA-blaM fusions covering the entire MexA polypeptide were constructed, and all fusion sites were shown to be located at the periplasm. To answer the question of whether or not membrane anchoring is essential for the MexA function, we replaced the signal sequence of the MexA protein with that of the azurin protein, which contains a cleavable signal sequence but no lipid modification site. The signal sequence of the azurin-MexA hybrid protein was properly processed and bore the mature MexA, which was fully recovered in the soluble fraction. The transformant, which expressed azurin-MexA hybrid protein restored the antibiotic resistance to a level indistinguishable from that of the wild-type strain. We concluded from these results that the MexA protein is fully functional as expressed in the periplasmic space without anchoring the inner membrane. This finding questioned the assumption that the membrane fusion proteins connect the inner and outer membranes.

Amino Acid Sequence↗
PubMed2005

Lipid-derived modifications of plasma proteins in experimental and human diabetes.

Plasma from two diabetic rat models and human diabetic patients was analyzed to investigate the hypothesis that enhanced oxidative stress in diabetes promotes lipid-derived protein modification. We evaluated the nonenzymatic modification of plasma protein by oxidized phospholipids, including measurement of protein-bound pentanedioate, nonanedioate, and hexanoate, all derived from oxidation of phospholipid polyunsaturated fatty acids. Generally pentanedioate was higher in diabetic compared with nondiabetic control groups, and nonanedioate was also higher in the diabetic rat models. We conclude that diabetes is associated with higher levels of phospholipid-derived protein modification in both animal models and human diabetes. Their role in the development of diabetes vascular complications warrants further research.

Animals↗
PubMed1995

Effect of ciclosporin on serum lipids and modification with LSL 90202, a lysine salt of eicosapentaenoic acid.

Ciclosporin (CS-A) has recently been considered a separate risk factor for the development of hyperlipidemia in transplant patients. In the present work, the effect of chronic CS-A administration on serum lipids and its modification using dietary supplementation with LSL 90202, a lysine salt of eicosapentaenoic acid, was studied. Thirty-one male Wistar rats were divided into four groups, receiving (1) 20 mg/kg CS-A in olive oil (CS-A group; n = 8); (2) isovolumetric olive oil (olive oil groups; n = 8); (3) 20 mg/kg CS-A in olive oil plus 20 mg/kg LSL 90202 (CS-A + LSL 20 group;) and (4) 20 mg/kg CS-A in olive oil plus 40 mg/kg LSL 90202 (CS-A+LSL 40 group; n = 8). Both, CS-A and LSL 90202 were given by daily gavage. On day 28, CS-A whole-blood levels and serum levels of total cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol fractions (HDL, HDL-2, HDL-3, non-HDL), and malondialdehyde were measured. On day 28, the rats given CS-A showed significantly higher cholesterol, triglyceride, and non-HDL cholesterol serum levels than rats given olive oil. Rats given CS-A and LSL 90202 (20 mg/kg) showed significantly lower triglyceride serum levels than rats given CS-A only. Rats given CS-A and LSL 90202 (40 mg/kg) showed significantly lower triglyceride, total cholesterol, and non-HDL cholesterol serum levels than rats given CS-A only. There were no differences in HDL, HDL-2, and HDL-3 cholesterol serum levels between the groups. The CS-A whole-blood levels were not different between groups of animals given CS-A.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Cytotoxicity of an anti-cancer lysophospholipid through selective modification of lipid raft composition.

Edelfosine is a prototypical member of the alkylphosphocholine class of antitumor drugs. Saccharomyces cerevisiae was used to screen for genes that modulate edelfosine cytotoxicity and identified sterol and sphingolipid pathways as relevant regulators. Edelfosine addition to yeast resulted in the selective partitioning of the essential plasma membrane protein Pma1p out of lipid rafts. Microscopic analysis revealed that Pma1p moved from the plasma membrane to intracellular punctate regions and finally localized to the vacuole. Consistent with altered sterol and sphingolipid synthesis resulting in increased edelfosine sensitivity, mislocalization of Pma1p was preceded by the movement of sterols out of the plasma membrane. Cells with enfeebled endocytosis and vacuolar protease activities prevented edelfosine-mediated (i) mobilization of sterols, (ii) loss of Pma1p from lipid rafts, and (iii) cell death. The activities of proteins and signaling processes are meaningfully altered by changes in lipid raft biophysical properties. This study points to a novel mode of action for an anti-cancer drug through modification of plasma membrane lipid composition resulting in the displacement of an essential protein from lipid rafts.

Antineoplastic Agents↗
PubMed2001

Lipid A modifications in polymyxin-resistant Salmonella typhimurium: PMRA-dependent 4-amino-4-deoxy-L-arabinose, and phosphoethanolamine incorporation.

Lipid A of Salmonella typhimurium can be resolved into multiple molecular species. Many of these substances are more polar than the predominant hexa-acylated lipid A 1,4'-bisphosphate of Escherichia coli K-12. By using new isolation methods, we have purified six lipid A subtypes (St1 to St6) from wild type S. typhimurium. We demonstrate that these lipid A variants are covalently modified with one or two 4-amino-4-deoxy-l-arabinose (l-Ara4N) moieties. Each lipid A species with a defined set of polar modifications can be further derivatized with a palmitoyl moiety and/or a 2-hydroxymyristoyl residue in place of the secondary myristoyl chain at position 3'. The unexpected finding that St5 and St6 contain two l-Ara4N residues accounts for the anomalous structures of lipid A precursors seen in S. typhimurium mutants defective in 3-deoxy-d-manno-octulosonic acid biosynthesis in which only the 1-phosphate group is modified with the l-Ara4N moiety (Strain, S. M., Armitage, I. M., Anderson, L., Takayama, K., Quershi, N., and Raetz, C. R. H. (1985) J. Biol. Chem. 260, 16089-16098). Phosphoethanolamine (pEtN)-modified lipid A species are much less abundant than l-Ara4N containing forms in wild type S. typhimurium grown in broth but accumulate to high levels when l-Ara4N synthesis is blocked in pmrA(C)pmrE(-) and pmrA(C)pmrF(-) mutants. Purification and analysis of selected compounds demonstrate that one or two pEtN moieties may be present. Our findings show that S. typhimurium contains versatile enzymes capable of modifying both the 1- and 4'-phosphates of lipid A with l-Ara4N and/or pEtN groups. PmrA null mutants of S. typhimurium produce lipid A species without any pEtN or l-Ara4N substituents. However, PmrA is not needed for the incorporation of 2-hydroxymyristate or palmitate.

Amino Sugars↗
PubMed1990

Enhanced activity of the tricarboxylate carrier and modification of lipids in hepatic mitochondria from hyperthyroid rats.

The effect of hyperthyroidism on the activity of the mitochondrial tricarboxylate carrier has been studied. The activity of this transporting system in liver mitochondria was quantitatively determined by the rate of malate-[14C]citrate exchange using the 1,2,3-benzene-tricarboxylate inhibitor stop technique. It has been found that the rate of citrate uptake is significantly enhanced in liver mitochondria from hyperthyroid rats as compared to that obtained in mitochondria from control rats. Kinetic analysis of the malate-citrate exchange reaction indicates that only the Vmax of this transporting process is enhanced, while there is practically no change in the Km values. Inhibitor titrations with the inhibitor palmitoyl-CoA show that mitochondria from hyperthyroid rats require the same concentrations of inhibitor to produce 100% inhibition of citrate uptake as control mitochondria, suggesting that the amount of functional translocase enzyme present is unaffected. The Arrhenius plot characteristics differ for tricarboxylate carrier activity in mitochondria from hyperthyroid rats as compared with control rats in that the break point of the biphasic plot decreases from 18.1 +/- 1.4 degrees C in controls to 12.9 +/- 1.2 degrees C in hyperthyroid animals. The hepatic mitochondrial lipid composition is altered significantly in hyperthyroid rats; the total cholesterol decreases and the phospholipids increase. The liver mitochondrial phospholipid composition is altered significantly in hyperthyroid rats. In particular negatively charged phospholipid cardiolipin increases by more than 50%. Minor alterations were found in the pattern of fatty acids. The thyroid hormone induced change in the activity of the tricarboxylate carrier can be ascribed either to a general modification of membrane lipid composition which increases the membrane fluidity and in turn the mobility of the carrier or to a more localized change of lipid domain (cardiolipin content) surrounding the carrier molecule in the mitochondrial membrane.

Animals↗
PubMed2001

Modification of lipid A biosynthesis in Neisseria meningitidis lpxL mutants: influence on lipopolysaccharide structure, toxicity, and adjuvant activity.

Two genes homologous to lpxL and lpxM from Escherichia coli and other gram-negative bacteria, which are involved in lipid A acyloxyacylation, were identified in Neisseria meningitidis strain H44/76 and insertionally inactivated. Analysis by tandem mass spectrometry showed that one of the resulting mutants, termed lpxL1, makes lipopolysaccharide (LPS) with penta- instead of hexa-acylated lipid A, in which the secondary lauroyl chain is specifically missing from the nonreducing end of the GlcN disaccharide. Insertional inactivation of the other (lpxL2) gene was not possible in wild-type strain H44/76 expressing full-length immunotype L3 lipopolysaccharide (LPS) but could be readily achieved in a galE mutant expressing a truncated oligosaccharide chain. Structural analysis of lpxL2 mutant lipid A showed a major tetra-acylated species lacking both secondary lauroyl chains and a minor penta-acylated species. The lpxL1 mutant LPS has retained adjuvant activity similar to wild-type meningococcal LPS when used for immunization of mice in combination with LPS-deficient outer membrane complexes from N. meningitidis but has reduced toxicity as measured in a tumor necrosis factor alpha induction assay with whole bacteria. In contrast, both adjuvant activity and toxicity of the lpxL2 mutant LPS are strongly reduced. As the combination of reduced toxicity and retained adjuvant activity has not been reported before for either lpxL or lpxM mutants from other bacterial species, our results demonstrate that modification of meningococcal lipid A biosynthesis can lead to novel LPS species more suitable for inclusion in human vaccines.

Acyltransferases↗
PubMed1993

Hypochlorous acid-mediated modification of cholesterol and phospholipid: analysis of reaction products by gas chromatography-mass spectrometry.

Oxidative modification of membrane lipids by hypochlorous acid could be an important element in the mechanism of membrane disruption by activated neutrophils. We have previously shown that hypochlorous acid reacts with unsaturated fatty acids of membrane phospholipids to give fatty acid chlorohydrins (Winterbourn et al. 1992. Arch. Biochem. Biophys. 296: 547-555). In the present study, we examined the reaction of cholesterol in bilayers with an inert phospholipid carrier. Product separation and identification was performed using gas chromatography-mass spectrometry after trimethylsilyl-derivatization. Unlike the reaction of hypochlorous acid with unsaturated fatty acids, no chlorohydrin derivatives were found with cholesterol. Instead, the main oxidation products were identified as the epimeric cholesterol 5,6-epoxides and 4-hydroxycholesterol, while several other hydroxy- and keto-derivatives were also found in smaller amounts. Analysis of the products obtained after reaction of vesicles composed of a mixture of several unsaturated phospholipid species plus cholesterol revealed that the individual fatty acids and cholesterol all exhibit comparable susceptibilities toward hypochlorous acid. Using myeloperoxidase to generate hypochlorous acid, basically the same products and product distribution were obtained. These studies show that unsaturated phospholipids and cholesterol can be profoundly modified by reaction with hypochlorous acid. This warrants further investigation to define the role of lipid modifications in neutrophil-mediated membrane disruption.

Cholesterol↗
PubMed1991

Differential effects of lecithin and cholesterol on the immunoreactivity and conformation of apolipoprotein A-I in high density lipoproteins.

Recently identified epitopes in apoA-I define a distinct N-terminal region with a complex tertiary structure, characterized by multiple discontinuous epitopes. Other epitopes are constituted of short domains centered either on beta-turns or random coils or on the 22-mer amphipathic alpha-helices (Marcel, Y. L., Provost, P. R., Koa, H., Raffaï, E., Vu Dac, N., Fruchart, J.-C., and Rassart, E. (1991) J. Biol. Chem. 266, 3644-3653). The compared immunoreactivity of seven epitopes studies here in response first to delipidation of high density lipoprotein (HDL) apoA-I by detergents, and second to modifications of HDL lipid composition by phospholipase A2 or by enrichment in surface lipids demonstrates that apoA-I has a flexible conformation which is readily responsive to the nature and concentration of bound lipids and that the structure of lipid-free apoA-I is significantly different from that of HDL-bound apoA-I, possibly representing a condensed molecule with several masked domains. In HDL apoA-I, these epitopes define five distinct domains which are characterized by particular responses to lipid modifications. However, two domains, each starting at the N-terminal beta-turn of an amphipathic alpha-helical repeat (residues 99-121 and 186-209, respectively) have almost identical immunoreactivity whether after detergent treatment or after changes in cholesterol and phospholipid levels, a property which probably reflects the known periodicity of apoA-I structural 22-mers. The immunoreactivity of a discontinuous epitope, representative of the N-terminal domain, is inversely related to the concentration of phospholipids, a unique characteristic among the epitopes tested here which indicates that the complex N-terminal region interacts with phospholipids, either directly or indirectly. These studies demonstrate that the conformation of multiple domains of HDL apoA-I is dependent on lipid phase composition and differentially affected by cholesterol and phospholipids.

Apolipoprotein A-I↗
PubMed2002

Fluorescence analysis of receptor-G protein interactions in cell membranes.

The dynamics of G protein heterotrimer complex formation and disassembly in response to nucleotide binding and receptor activation govern the rate of responses to external stimuli. We use a novel flow cytometry approach to study the effects of lipid modification, isoform specificity, lipid environment, and receptor stimulation on the affinity and kinetics of G protein subunit binding. Fluorescein-labeled myristoylated Galpha(i1) (F-alpha(i1)) was used as the ligand bound to Gbetagamma in competition binding studies with differently modified Galpha subunit isoforms. In detergent solutions, the binding affinity of Galpha(i) to betagamma was 2 orders of magnitude higher than for Galpha(o) and Galpha(s) (IC50 of 0.2 nM vs 17 and 27 nM, respectively), while in reconstituted bovine brain lipid vesicles, binding was slightly weaker. The effects of receptor on the G protein complex were assessed in alpha(2A)AR receptor expressing CHO cell membranes into which purified betagamma subunits and F-alpha(i1) were reconstituted. These cell membrane studies led to the following observations: (1) binding of alpha subunit to the betagamma was not enhanced by receptor in the presence or absence of agonist, indicating that betagamma contributed essentially all of the binding energy for alpha(i1) interaction with the membrane; (2) activation of the receptor facilitated GTPgammaS-stimulated detachment of F-alpha(i1) from betagamma and the membrane. Thus flow cytometry permits quantiatitive and real-time assessments of protein-protein interactions in complex membrane environments.

Adrenergic alpha-2 Receptor Agonists↗