Thromboxane A2 signalling in humans: a 'Tail' of two receptors.

Since its discovery in 1975, we now have a wealth of knowledge relating to the biochemical, pharmacological and physiological actions of thromboxane A(2) (TXA(2)) and its related metabolites. These molecular insights have been greatly expedited by the molecular cloning and characterization of a cDNA for the human TXA(2) receptor, now termed the T Prostanoid or TP receptor, from a megakaryocytic/placental cDNA library in 1991, and later through the discovery of a cDNA encoding a second isoform of the human TP receptor in 1994. The requirement for two TP receptors in primates, but not in other species thus far investigated, is unclear, but points to potential species-specific physiological differences. In this review, I will describe some recent advances in the research field of TXA(2)/TP receptor signalling, focusing particularly on studies pertaining to the human TP receptor isoforms.

Protein kinase A-mediated phosphorylation of serine 357 of the mouse prostacyclin receptor regulates its coupling to G(s)-, to G(i)-, and to G(q)-coupled effector signaling.

The prostacyclin receptor (IP) is primarily coupled to G alpha(s)-dependent activation of adenylyl cyclase; however, a number of studies indicate that the IP may couple to other secondary effector systems perhaps in a species-specific manner. In the current study, we investigated the specificity of G protein:effector coupling by the mouse (m) IP overexpressed in human embryonic kidney 293 cells and endogenously expressed in murine erythroleukemia cells. The mIP exhibited efficient G alpha(s) coupling and concentration-dependent increases in cAMP generation in response to the IP agonist cicaprost; however, mIP also coupled to G alpha(i) decreasing the levels of cAMP in forskolin-treated cells. mIP coupling to G alpha(i) was pertussis toxin-sensitive and was dependent on protein kinase (PK) A activation status. In addition, the mIP coupled to phospholipase C (PLC) activation in a pertussis toxin-insensitive, G alpha(i)-, G beta gamma-, and PKC-independent but in a G alpha(q)- and PKA-dependent manner. Whole cell phosphorylation assays demonstrated that the mIP undergoes cicaprost-induced PKA phosphorylation. mIP(S357A), a site-directed mutant of mIP, efficiently coupled to G alpha(s) but failed to couple to G alpha(i) or to efficiently couple to G alpha(q):PLC. Moreover, mIP(S357A) did not undergo cicaprost-induced phosphorylation confirming that Ser(357) is the target residue for PKA-dependent phosphorylation. Finally, co-precipitation experiments permitted the detection of G alpha(s), G alpha(i), and G alpha(q) in the immunoprecipitates of mIP, whereas only G alpha(s) was co-precipitated with mIP(S357A) indicating that Ser(357) of mIP is essential for G alpha(i) and G alpha(q) interaction. Moreover, inhibition of PKA blocked co-precipitation of mIP with G alpha(i) or G alpha(q). Taken together our data indicate that the mIP, in addition to coupling to G alpha(s), couples to G alpha(i) and G alpha(q); however, G alpha(i) and G alpha(q) coupling is dependent on initial cicaprost-induced mIP:G alpha(s) coupling and phosphorylation of mIP by cAMP-dependent PKA where Ser(357) was identified as the target residue for PKA phosphorylation.

Prostaglandin D(2) receptor-mediated desensitization of the alpha isoform of the human thromboxane A(2) receptor.

Thromboxane (TX) A(2) and prostaglandin (PG) D(2) mediate opposing actions in platelets and in vascular and non-vascular smooth muscle. Here, we investigated the effects of stimulation of the PGD(2) receptor (DP) on signaling by the TXA(2) receptor (TP) expressed in human platelets and in human embryonic kidney (HEK) 293 cells over-expressing the individual TP alpha and TP beta isoforms. In platelets, the selective DP agonist BW245C abolished TP-mediated mobilization of intracellular calcium ([Ca(2+)](i)) and inhibited platelet aggregation in response to the TXA(2) mimetic U46619. DP-mediated desensitization of TP signaling in platelets was prevented by pretreatment with the cAMP-dependent PKA inhibitor, H-89, but was unaffected by the PKC inhibitor GF 109203X. In HEK 293 cells, signaling by TP alpha, but not TP beta, was subject to DP-mediated desensitization in a PKA-dependent, PKC-independent manner. U46619-induced signaling by TP(Delta 328), a truncated variant of TP containing only those residues common to TP alpha and TP beta, was insensitive to prior DP stimulation, indicating that the carboxyl terminal tail of TPalpha contains the target site(s) for DP-mediated desensitization. Mutation of Ser(329) to Ala(329) within a consensus PKA site in TP alpha rendered the mutant TP alpha(S329A) insensitive to BW245C-mediated desensitization. Whole cell phosphorylation assays established that TP alpha, but not TP beta or TP alpha(S329A), was subject to DP-mediated phosphorylation and that TP alpha phosphorylation was blocked by the PKA inhibitor H-89. These data establish that TP alpha, but not TP beta, is subject to DP-mediated cross desensitization, which occurs through direct PKA-mediated phosphorylation of TP alpha at Ser(329).

Thromboxane A(2) receptor mediated activation of the mitogen activated protein kinase cascades in human uterine smooth muscle cells.

Both thromboxane (TX) A(2) and 8-epi prostaglandin (PG) F(2alpha) have been reported to stimulate mitogenesis of vascular smooth muscle (SM) in a number of species. However, TXA(2) and 8-epiPGF(2alpha) mediated mitogenic signalling has not been studied in detail in human vascular SM. Thus, using the human uterine ULTR cell line as a model, we investigated TXA(2) receptor (TP) mediated mitogenic signalling in cultured human vascular SMCs. Both the TP agonist U46619 and 8-epiPGF(2alpha) elicited time and concentration dependent activation of the extracellular signal regulated kinase (ERK)s and c-Jun N-terminal kinase (JNK)s in ULTR cells. Whereas the TP antagonist SQ29548 abolished U46619 mediated signalling, it only partially inhibited 8-epiPGF(2alpha) mediated ERK and JNK activation in ULTR cells. Both U46619 and 8-epiPGF(2alpha) induced ERK activations were inhibited by the protein kinase (PK) C, PKA and phosphoinositide 3-kinase inhibitors GF109203X, H-89 and wortmannin, respectively, but were unaffected by pertussis toxin. In addition, U46619 mediated ERK activation in ULTR cells involves transactivation of the epidermal growth factor (EGF) receptor. In humans, TXA(2) signals through two distinct TP isoforms. In investigating the involvement of the TP isoforms in mitogenic signalling, both TPalpha and TPbeta independently directed U46619 and 8-epiPGF(2alpha) mediated ERK and JNK activation in human embryonic kidney (HEK) 293 cells over-expressing the individual TP isoforms. However, in contrast to that which occurred in ULTR cells, SQ29548 abolished 8-epiPGF(2alpha) mediated ERK and JNK activation through both TPalpha and TPbeta in HEK 293 cells providing further evidence that 8-epiPGF(2alpha) may signal through alternative receptors, in addition to the TPs, in human uterine ULTR cells.

The effects of the statins lovastatin and cerivastatin on signalling by the prostanoid IP-receptor.

The prostanoid-IP receptor may be unique among G protein coupled receptors in that it is isoprenylated. In this study, we investigated the effects of the statins lovastatin and cerivastatin on signalling by the mouse (m) IP and the human (h) IP receptors, over-expressed in human embryonic kidney (HEK) 293 cells and by the hIP receptor, endogenously expressed in human erythroleukaemia cells. Both statins significantly reduced IP receptor-mediated cyclic AMP generation and intracellular calcium ([Ca(2+)](i)) mobilization in a time and concentration dependent manner but had no effect on signalling by the non-isoprenylated beta(2) adrenergic receptor or by the human prostanoid-TP receptor isoforms. Cerivastatin (IC(50), 50 - 90 nM) was significantly more potent than lovastatin (IC(50), 0.80 - 4.2 microM) in inhibiting IP receptor signalling. Whereas IC(50) values indicated that the hIP receptor was significantly more sensitive than the mIP receptor to the statins, the extent of inhibition of cyclic AMP generation by the mIP receptor was significantly greater than that of the hIP receptor to either statin, even at the highest concentrations used. Pretreatment with either statin significantly reduced IP receptor mediated desensitization of signalling by the h.TPalpha, but not by the h.TPbeta, receptor isoform. These data generated in whole cells point to the possibility that statin therapy may interfere with IP receptor signalling in vivo; such interference may be extenuated under conditions where circulating statin levels are elevated and may account, in part, for some of the pleiotropic affects of the statins not attributed solely to their lipid lowering properties.

Regulation of the human prostanoid TPalpha and TPbeta receptor isoforms mediated through activation of the EP(1) and IP receptors.

The intermolecular cross-regulation mediated by the prostanoid IP-receptor (IP)/EP(1) receptor (EP(1)) agonists PGI(2) and 17 phenyl trinor PGE(2) on TP receptor (TP) signalling within platelets was compared to that which occurs to the individual TPalpha and TPbeta receptors over-expressed in human embryonic kidney (HEK) 293 cells. Ligand mediated TP receptor activation was monitored by analysing mobilization of intracellular calcium ([Ca(2+)](i)) following stimulation with the selective thromboxane (TX) A(2) mimetic U46619. Consistent with previous studies, in platelets, PGI(2) acting through endogenous IP receptors completely inhibited U46619-mediated TP receptor signalling in a protein kinase (PK) A-dependent, PKC-independent manner. In HEK 293 cells, PGI(2), acting through endogenous AH6809 sensitive EP(1) rather than IP receptors, and the selective EP(1) receptor agonist 17 phenyl trinor PGE(2) antagonized U46619-mediated signalling by both TPalpha and TPbeta receptors in a PKC-dependent, PKA-independent manner. The maximum response induced by either ligand was significantly (P<0.005) greater for the TPalpha receptor than the TPbeta receptor, pointing to possible physiologic differences between the TP isoforms, although the potency of each ligand was similar for both TP receptors. TP(Delta328), a truncated variant of TP receptor lacking the C-tail sequences unique to TPalpha or TPbeta receptors, was not sensitive to EP(1) receptor-mediated regulation by PGI(2) or 17 phenyl trinor PGE(2) In conclusion, these data confirm that TPalpha and TPbeta receptors are subject to cross regulation by EP(1) receptor signalling in HEK 293 cells mediated by PKC at sites unique to the individual TP receptors and that TPalpha receptor responses are significantly more reduced by EP(1) receptor regulation than those of the TPbeta receptor.

The alpha, but not the beta, isoform of the human thromboxane A2 receptor is a target for prostacyclin-mediated desensitization.

In this study, we examined the effects the prostacyclin receptor (IP) agonist cicaprost exhibited on U46619-mediated thromboxane A(2) receptor (TP) signaling in platelets and compared it to that which occurs in human embryonic kidney (HEK) 293 cells stably overexpressing the individual TPalpha or TPbeta isoforms. Consistent with previous studies, cicaprost abrogated U46619-mediated platelet aggregation and mobilization of intracellular calcium ([Ca(2+)](i)). In HEK 293 cells, signaling by TPalpha, but not TPbeta, was subject to IP-mediated desensitization in a protein kinase A-dependent, protein kinase C-independent manner. Desensitization of TPalpha signaling was independent of the nature of the IP agonist used, the level of IP expression, or the subtype of G(q) protein. Signaling by TP(Delta)(328), a truncated variant of TP devoid of the divergent residues of the TPs, or by TPalpha(S329A), a site-directed mutant of TPalpha, were insensitive to IP agonist activation. Whole cell phosphorylations established that TPalpha, but not TPbeta or TPalpha(S329A), is subject to IP-mediated phosphorylation and that TPalpha phosphorylation is inhibited by H-89. Thus, we conclude that TPalpha, but not TPbeta, is subject to cross-desensitization by IP mediated through direct protein kinase A phosphorylation at Ser(329) and propose that TPalpha may be the isoform physiologically relevant to TP:IP-mediated vascular hemostasis.

Investigation of the role of the carboxyl-terminal tails of the alpha and beta isoforms of the human thromboxane A(2) receptor (TP) in mediating receptor:effector coupling.

We have investigated the functional coupling of alpha and beta isoforms of the human thromboxane A(2) receptor (TP) to Galpha(16) and Galpha(12) members of the G(q) and G(12) families of heterotrimeric G proteins in human embryonic kidney (HEK) 293 cell lines HEK.alpha10 or HEK.beta3, stably over-expressing TPalpha and TPbeta, respectively. Moreover, using HEK.TP(Delta328) cells which over-express a variant of TP truncated at the point of divergence of TPalpha and TPbeta, we investigated the requirement of the C-tail per se in mediating G protein coupling and effector activation. Both TPalpha and TPbeta couple similarly to Galpha(16) to affect increases in inositol 1,4,5-trisphosphate (IP(3)) and mobilisation of intracellular calcium ([Ca(2+)](i)) in response to the TP agonist U46619. Whilst both TP isoforms mediated [Ca(2+)](i) mobilisation in cells co-transfected with Galpha(12), neither receptor generated corresponding increases in IP(3), indicating that the Galpha(12)-mediated increases in [Ca(2+)](i) do not involve PLC activation. Verapamil, an inhibitor of voltage dependent Ca(2+) channels, reduced [Ca(2+)](i) mobilisation in TPalpha and TPbeta cells co-transfected with Galpha(12) to approximately 40% of that mobilised in its absence, whereas [8-(N,N-diethylamino)-octyl-3,4, 5-trimethoxybenzoate, hydrochloride] (TMB-8), an antagonist of intracellular Ca(2+) release, had no effect on [Ca(2+)](i) mobilisation by either receptor isoform co-transfected with Galpha(12). Despite the lack of differential coupling specificity by TPalpha and TPbeta, TP(Delta328) signalled more efficiently in the absence of a co-transfected G protein compared to the wild type receptors but, on the other hand, displayed an impaired ability to couple to co-transfected Galpha(11), Galpha(12) or Galpha(16) subunits. In studies investigating the role of the C-tail in influencing coupling to the effector adenylyl cyclase, similar to TPalpha but not TPbeta, TP(Delta328) coupled to Galpha(s), leading to increased adenosine 3',5'-cyclic monophosphate (cAMP), rather than to Galpha(i). Whereas TP(Delta328) signalled more efficiently in the absence of co-transfected G protein compared to the wild type TPalpha, co-transfection of Galpha(s) did not augment cAMP generation by TP(Delta328). Hence, from these studies involving the wild type TPalpha, TPbeta and TP(Delta328), we conclude that the C-tail sequences of TP are not a major determinant of G protein coupling specificity to Galpha(11) and Galpha(16) members of the G(q) family or to Galpha(12); it may play a role in determining G(s) versus G(i) coupling and may act as a determinant of coupling efficiency.

The prostacyclin receptor is isoprenylated. Isoprenylation is required for efficient receptor-effector coupling.

The prostacyclin receptor (IP), a G protein-coupled receptor, mediates the actions of the prostanoid prostacyclin and its mimetics. IPs from a number of species each contain identically conserved putative isoprenylation CAAX motifs, each with the sequence CSLC. Metabolic labeling of human embryonic kidney (HEK) 293 cells stably overexpressing the hemagluttinin epitope-tagged IP in the presence of [(3)H]mevalonolactone established that the mouse IP is isoprenylated. Studies involving in vitro assays confirmed that recombinant forms of the human and mouse IP are modified by carbon 15 farnesyl isoprenoids. Disruption of isoprenylation, by site-directed mutagenesis of Cys(414) to Ser(414), within the CAAX motif, abolished isoprenylation of IP(SSLC) both in vitro and in transfected cells. Scatchard analysis of the wild type (IP) and mutant (IP(SSLC)) receptor confirmed that each receptor exhibited high and low affinity binding sites for [(3)H]iloprost, which were not influenced by receptor isoprenylation. Whereas stable cell lines overexpressing IP generated significant agonist (iloprost and cicaprost)-mediated increases in cAMP relative to nontransfected cells, cAMP generation by IP(SSLC) cells was not significantly different from the control, nontransfected HEK 293 cells. Moreover, co-expression of the alpha (alpha) subunit of Gs generated significant augmentations in cAMP by IP but not by IP(SSLC) cells. Whereas IP also demonstrated significant, dose-dependent increases in [Ca(2+)](i) in response to iloprost or cicaprost compared with the nontransfected HEK 293 cells, mobilization of [Ca(2+)](i) by IP(SSLC) was significantly impaired. Co-transfection of cells with either Galpha(q) or Galpha(11) resulted in significant augmentation of agonist-mediated [Ca(2+)](i) mobilization by IP cells but not by IP(SSLC) cells or by the control, HEK 293 cells. In addition, inhibition of isoprenylation by lovastatin treatment significantly reduced agonist-mediated cAMP generation by IP in comparison to the nonisoprenylated beta(2) adrenergic receptor or nontreated cells. Hence, isoprenylation of IP does not influence ligand binding but is required for efficient coupling to the effectors adenylyl cyclase and phospholipase C.

Expression and tissue distribution of the mRNAs encoding the human thromboxane A2 receptor (TP) alpha and beta isoforms.

The human thromboxane A2 receptor (TP), a G protein-coupled receptor, exists as two isoforms, TPalpha and TPbeta, which arise by alternative mRNA splicing and differ exclusively in their carboxyl terminal cytoplasmic regions. In this study, a reverse transcriptase-polymerase chain reaction (RT-PCR)-based strategy was developed to examine the expression of the TPs in tissues of physiologic relevance to TXA2. Although most of the 17 different cell/tissue types examined expressed both TP isoforms, the liver hepatoblastoma HepG2 cell line was found to exclusively express TPalpha mRNA. In most cell types, TPalpha mRNA predominated over TPbeta mRNA. Moreover, although the levels of TPalpha mRNA expression were similar in most of the cell/tissue types examined, extensive differences in the levels of TPbeta mRNA were observed. Consequently, the relative expression of TPalpha: TPbeta mRNA varied considerably due to extensive differences in TPbeta mRNA expression. Most strikingly, primary HUVECs were found to express: (i) low levels of TPbeta and (ii) approximately 6-fold greater levels of TPalpha than TPbeta. These data were confirmed in the spontaneously transformed HUVEC derived ECV304 cell line. Expression of TP mRNAs in the various tissue/cells correlated with protein expression, as assessed by radioligand binding using the selective TP antagonist [3H]SQ29,548.

Characterization of the role of N-linked glycosylation on the cell signaling and expression of the human thromboxane A2 receptor alpha and beta isoforms.

The alpha and beta isoforms of the thromboxane A2 receptor (TP) mediate the actions of the prostanoid thromboxane A2 and its mimetics in humans. The amino terminal region of the TPs contains two consensus N-linked glycosylation sites at asparagine (N) residues N4 and N16. In this study, we explored the significance of N-linked glycosylation on the signaling and surface expression of the human TP isoforms. Inhibition of N-linked glycosylation reduced selective radioligand ([3H]SQ29,548) binding by either TP in both human erythroleukemia cells and in transfected human embryonic kidney 293 cells. Moreover, site-directed mutagenesis of the putative glycosylation sites of TPalpha revealed that radioligand binding also was reduced greatly for both the single (TPalphaN4-Q4, TPalphaN16-Q16) and double (TPalphaN4,N16-Q4,Q16) mutants, yielding levels of 8% binding relative to the wild-type TPalpha for the double mutants. Reductions in ligand binding were caused by decreased maximal binding and not by changes in affinity (Kd) or in specificity of the receptors for [3H]SQ29,548 or other ligands. Subcellular fractionation confirmed that, in relation to total TP expression, membrane expression was not altered in TPalphaN4-Q4 or TPalphaN16-Q16 but was reduced to levels of 55% of total expression in TPalphaN4,Q4-N16,Q16. Inhibition of glycosylation reduced, but did not abolish, agonist (U46619) mediated intracellular Ca++ mobilization by TPalpha or TPbeta and cAMP production by TPalpha. Thus, N-linked glycosylation of the human TP isoforms is important for ligand binding, efficient second messenger signaling and efficient membrane expression.

The human thromboxane A2 receptor alpha isoform (TP alpha) functionally couples to the G proteins Gq and G11 in vivo and is activated by the isoprostane 8-epi prostaglandin F2 alpha.

To establish whether the thromboxane A2 (TXA2) receptor (TP) functionally couples to the Gq family of heterotrimeric G proteins in vivo, we have coexpressed the cDNAs coding for the human platelet/placental TP alpha isoform (TP alpha) and the alpha subunits of Gq or G11 in human embryonic kidney (HEK) 293 cells. TP activation in response to ligand stimulation was monitored by analyzing mobilization of intracellular calcium (Ca++i) in FURA2/AM-loaded transfected HEK 293 and in platelets. Second, we wished to examine the possible interaction of the isoprostane 8-epi prostaglandin F2 alpha with the TP alpha, in transfected HEK 293 cells and with the TPs expressed in platelets. Thus both the prostaglandin endoperoxide/TXA2 analog (U46619) and the 8-epi PGF2 alpha were utilized as ligand probes of TP alpha activation. The results demonstrate that each ligand induced elevations of Ca++i levels in HEK 293 cells, cotransfected with either the TP alpha and G alpha q or the TP alpha and G alpha 11, and also in platelets. Initial stimulation of these cells with U46619 or 8-epi PGF 2 alpha desensitized a subsequent rise in [Ca++]i in response to U46619 or 8-epi PGF 2 alpha, respectively. Moreover, prestimulation with U46619 desensitized a subsequent rise in Ca++i concentration in response to 8-epi PGF 2 alpha, and vice versa. These responses were blocked by the TP antagonist SQ29,548 in both cell types. In contrast, prestimulation of the transfected HEK 293 cells or platelets with thrombin did not desensitize a subsequent rise in [Ca++]i in response to U46619 or 8-epi PGF 2 alpha. After stimulation with either U46619 or 8-epi PGF 2 alpha, no significant rise in Ca++i levels was observed in HEK 293 cells transfected with the TP alpha receptor only or in control cells transfected with the vector pCMV5. These results demonstrate that the TP alpha isoform functionally couples with either Gq or G11 in vivo, whether activated by a PG/TXA2 analog or by the F2 isoprostane 8-epi PGF2 alpha.

Phosphorylation and regulated expression of the human thromboxane A2 receptor.

Recombinant forms of the human thromboxane A2 (TXA2) receptor composed of the carboxyl-terminal amino acid residues 220-343 were phosphorylated in vitro by both cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC), and these phosphorylations were competed for by synthetic peptides corresponding to the proposed carboxyl-terminal cytoplasmic tail and/or the third extracellular loop of the receptor, respectively. Exogenous addition of PKA or PKC to membrane preparations of human embryonic kidney 293 cells, transfected with the TXA2 receptor, typically reduced TXA2 receptor binding by 10 and 30%, respectively. In vivo inhibition of PKC or PKA in the transfected human embryonic kidney 293 cells increased TXA2 receptor binding to 121.4% (+/- 5.3%) and 110.4% (+/- 4.6%), respectively, relative to control cells. In vivo activation of PKC in the platelet-like human erythroleukemia (HEL) cells by the phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in an initial reduction followed by a time-dependent increase in TXA2 receptor ligand binding. Stimulation of HEL cells with the TXA2 receptor agonist [15-(alpha,2 beta(5Z)-3 alpha(E,3S)-4 alpha)]-7-[3- (3-hydroxy-4-(p-iodophenoxy)-1-butenyl)-7-oxabicyclo-[-2.2,1-]hept -2-yl]- 5-heptenoic acid or basic fibroblast growth factor, alone or together, resulted in a marked decrease in TXA2 receptor binding. Northern blot studies in HEL cells demonstrated that PMA stimulation induced the expression of the TXA2 receptor gene with mRNA levels peaking following PMA stimulation for 4-8 h. This induction is consistent with the presence of a phorbol ester response element in promoter I of the TXA2 receptor gene. Dexamethasone did not induce the expression of the receptor gene, despite the presence of a glucocorticoid response element in promoter II of the TXA2 receptor gene. In summary, our results indicate that the cellular responses to TXA2 are mediated both by phosphorylation of the TXA2 receptor by different protein kinases and by regulated expression of the TXA2 receptor gene.

Cellular activation of thromboxane receptors.

Thromboxane A2 is an abundant and potent product of arachidonic acid metabolism in human platelets. Its clinical importance is highlighted by the efficacy of aspirin, which, due to its irreversible inhibition of the enzyme PGG/H synthase, selects the anucleate platelet as a particular target for extended duration of action. A single thromboxane receptor gene has been identified by southern blot; sequence polymorphism in the gene sequence has been identified. The recombinant receptor is also subject to posttranslational modifications, which may modify its affinity for natural ligands. Pharmacological studies have suggested some heterogeneity among thromboxane receptors. These observations have been rendered more interesting by the discovery of an F prostaglandin isomer, 8-epi-PGF2 alpha, which exerts its biological effects through a thromboxane (or closely related) receptor. This isomer can be generated in a free radical-catalyzed or cyclooxygenase-dependent manner.

Lipid modification of G proteins.

The heterotrimeric guanine nucleotide-binding G proteins, composed of α, ß, and γ subunits, act as signal transducers between cell surface receptors and downstream effector molecules, leading to changes in intracellular second messengers. The superfamily of ras-related low-molecular-mass GTP-binding G proteins is involved in a number of cellular functions, including cell differentiation and growth control, actin polymerization and cytoskeleton arrangement, and intracellular vesicular transport. The heterotrimeric G proteins and the ras-related low-molecular-mass G proteins are modified in vivo by a number of lipid groups, including palmitate, myristate, heterogeneous fatty-acyl groups (C12:0, C14:1, or C14:2 fatty-acyl groups), and C15 farnesyl or C20 geranylgeranyl isoprenoids. Lipid modification of G proteins increases the hydrophobicity of the proteins. In this review, we describe the various types of lipid modification of G proteins and discuss the significance of lipid modification with respect to G-protein function.

Transcription from the rat 45S ribosomal DNA promoter does not require the factor UBF.

For efficient transcription from the rat ribosomal DNA (rDNA) promoter by RNA polymerase I in vitro, at least two transcription factors, rat UBF and rat SL-1, are required. Transcription cannot take place in vitro in the absence of SL-1. On the other hand, there is considerable difference of opinion concerning the necessity for UBF in in vitro transcription mediated by RNA polymerase 1, and the requirement for UBF is not clear. Mammalian cells code for UBF1 and UBF2, two forms of UBF that differ in HMG box-2, one of four HMG boxes or DNA-binding domains. We have used a monospecific antibody raised to recombinant rat UBF to determine whether UBF1 and UBF2 are required for RNA polymerase I-mediated transcription. This antibody can detect as little as 1.35 x 10(-15) moles of UBF1 or UBF2 in an immunoblot. Fractionated extracts that were competent for transcription had no detectable UBF1 or UBF2 when assayed in immunoblots with this antiserum. This evidence supports the hypothesis that UBF is not required for transcription of the rat rDNA promoter in vitro and most likely functions as an auxillary transcription factor. In addition, we have fractionated rat UBF1 from UBF2 and tested each of them in in vitro transcription assays in which the 45S or spacer rDNA promoter template is limiting. UBF1 can activate transcription from either the 45S or spacer promoter under these conditions, whereas UBF2 cannot. This implies that there is a functional difference in the transactivation of RNA polymerase I by UBF1 and UBF2 in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of Rac 2. A cytosolic GTP-binding protein that regulates human neutrophil NADPH oxidase.

Human neutrophils and other phagocytes generate superoxide anion (O2-) as a means of destroying ingested microorganisms. O2- is produced by an NADPH-consuming oxidase composed of membrane and cytosolic components. Activation of the NADPH oxidase is absolutely dependent upon GTP, indicating the requirement for a GTP-binding protein in this process. We have utilized a five-step chromatographic procedure to isolate a GTP-binding protein from human neutrophil cytosol which can stimulate NADPH oxidase activity in a cell-free assay. Oxidase enhancing activity was shown to coisolate with this GTP-binding component, which was purified to apparent homogeneity. The GTP-binding protein was identified as Rac 2 by immunological reactivity and amino acid sequencing. Thus, Rac 2 appears to be a third cytosolic component required for human neutrophil NADPH oxidase activation. Recombinant Rac 2 was shown to bind guanine nucleotides in a Mg(2+)-dependent fashion. GDP dissociation rates were determined and shown to be regulated by the free Mg2+ concentration. Rac 2 was found to possess the highest rate of intrinsic GTP hydrolysis of any of the characterized members of the Ras superfamily. The biochemical properties of Rac 2 indicate it is likely to be subject to regulatory cofactors in vivo.

rab GTP-binding proteins with three different carboxyl-terminal cysteine motifs are modified in vivo by 20-carbon isoprenoids.

p21ras and several other ras-related GTP-binding proteins are modified post-translationally by addition of 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoids to cysteines within a conserved carboxyl-terminal sequence motif, Caa(M/S/L), where a is an aliphatic amino acid. Proteins ending with M or S are substrates for farnesyltransferase, whereas those ending with L are modified preferentially by geranylgeranyltransferase. We recently reported that GTP-binding proteins encoded by rab1B (GGCC), rab2 (GGCC), and rab5 (CCSN) are modified by 20-carbon isoprenyl derivatives of [3H]mevalonate when translated in vitro, despite having carboxyl-terminal sequences distinct from the Caa(M/S/L) motif. We now show that these proteins function as specific acceptors for geranylgeranyl in vitro and are modified by 20-carbon isoprenyl groups in COS cells metabolically labeled with [3H]mevalonate. Proteins encoded by rab4 and rab6, with yet another distinct carboxyl-terminal motif (xCxC), are similarly modified by 20-carbon isoprenoids in vitro and in vivo. The geranylgeranyl modification of rab5 protein (CCSN) is catalyzed by an enzyme in brain cytosol but not by a purified geranylgeranyltransferase that modifies GTP-binding proteins with the CaaL motif. Unlike the prenylation of proteins with Caa(M/S/L) termini, the prenylation of rab5 protein is not inhibited by a synthetic peptide based on its carboxyl-terminal sequence (TRNQCCSN). When cellular isoprenoid synthesis is blocked by treatment of cells with lovastatin, rab proteins that are normally localized in membranes of the endoplasmic reticulum, Golgi apparatus, and endosomes accumulate in the cytosol. This change in rab protein localization is reversed by providing cells with mevalonate. These findings suggest that geranylgeranyl modification underlies the ability of rab GTP-binding proteins to associate with intracellular membranes, where they are postulated to function as mediators of vesicular traffic.

Primary structure and processing of the Candida tsukubaensis alpha-glucosidase. Homology with the rabbit intestinal sucrase-isomaltase complex and human lysosomal alpha-glucosidase.

The nucleotide sequence of a 4.39-kb DNA fragment encoding the alpha-glucosidase gene of Candida tsukubaensis is reported. The cloned gene contains a major open reading frame (ORF 1) which encodes the alpha-glucosidase as a single precursor polypeptide of 1070 amino acids with a predicted molecular mass of 119 kDa. N-terminal amino acid sequence analysis of the individual subunits of the purified enzyme, expressed in the recombinant host Saccharomyces cerevisiae, confirmed that the alpha-glucosidase precursor is proteolytically processed by removal of an N-terminal signal peptide to yield the two peptide subunits 1 and 2, of molecular masses 63-65 kDa and 50-52 kDa, respectively. Both subunits are secreted by the heterologous host S. cerevisiae in a glycosylated form. Coincident with its efficient expression in the heterologous host, the C. tsukubaensis alpha-glucosidase gene contains many of the canonical features of highly expressed S. cerevisiae genes. There is considerable sequence similarity between C. tsukubaensis alpha-glucosidase, the rabbit sucrase-isomaltase complex (proSI) and human lysosomal acid alpha-glucosidase. The cloned DNA fragment from C. tsukubaensis contains a second open reading frame (ORF 2) which has the capacity to encode a polypeptide of 170 amino acids. The function and identity of the polypeptide encoded by ORF 2 is not known.