The critical cell signals that trigger cardiac hypertrophy and regulate the transition to heart failure are not known. To determine the role of G␣q-mediated signaling pathways in these events, transgenic mice were constructed that overexpressed wild-type G␣q in the heart using the ␣-myosin heavy chain promoter. Two-fold overexpression of G␣q showed no detectable effects, whereas 4-fold overexpression resulted in increased heart weight and myocyte size along with marked increases in atrial naturietic factor (Ϸ55-fold), ␤-myosin heavy chain (Ϸ8-fold), and ␣-skeletal actin (Ϸ8-fold) expression, and decreased (Ϸ3-fold) ␤-adrenergic receptor-stimulated adenylyl cyclase activity. All of these signals have been considered markers of hypertrophy or failure in other experimental systems or human heart failure. Echocardiography and in vivo cardiac hemodynamic studies indeed revealed impaired intrinsic contractility manifested as decreased fractional shortening (19 ؎ 2% vs. 41 ؎ 3%), dP͞dt max, a negative force-frequency response, an altered Starling relationship, and blunted contractile responses to the ␤-adrenergic agonist dobutamine. At higher levels of G␣q overexpression, frank cardiac decompensation occurred in 3 of 6 animals with development of biventricular failure, pulmonary congestion, and death. The element within the pathway that appeared to be critical for these events was activation of protein kinase C. Interestingly, mitogen-activated protein kinase, which is postulated by some to be important in the hypertrophy program, was not activated. The G␣q overexpressor exhibits a biochemical and physiologic phenotype resembling both the compensated and decompensated phases of human cardiac hypertrophy and suggests a common mechanism for their pathogenesis.

Expression of platelet thromboxane receptors is transcriptionally increased during megakaryocytic differentiation stimulated by phorbol 12-myristate 13-acetate (PMA). We previously cloned and characterized the promoter region of the human thromboxane receptor gene and localized PMA-responsive elements to a region between 1.84 and 1.95 kilobase pairs (kb) 5 of the transcription initiation site (D'Angelo, D. D., Davis, M. G., Houser, W. A., Eubank, J. J., Ritchie, M. E., and Dorn, G. W., II (1995) Circ. Res. 77, 466 -474). Herein we report the localization of the PMA response element to a 14-nucleotide C-rich sequence, flanked by an octanucleotide inverted repeat, located ؊1.938 to ؊1.925 kb 5 of the transcription start site of this gene. We further identify the PMA-responsive enhancer factor that binds to this Crich sequence as Sp1. Heterologous thromboxane receptor gene promoter/thymidilate kinase reporter constructs transfected into K562 cells exhibited PMA responsiveness when the C-rich element was included with additional 3 sequence from ؊1.924 to ؊1.84 kb. However, mutations of the C-rich element that disrupted a GC box located on the inverse strand eliminated PMA responsiveness and, in gel mobility shift assays, eliminated binding of Sp1. PMA treatment of K562 cells significantly increased, by 5-fold, Sp1 binding to the C-rich element and increased both phosphorylated and nonphosphorylated Sp1 protein levels by 2-fold. Furthermore, PMA treatment transiently increased Sp1 mRNA levels prior to increasing thromboxane receptor mRNA, suggesting that up-regulation of Sp1 contributes to up-regulation of thromboxane receptors. Finally, we have detected an unidentified K562 nuclear protein that binds specifically to the sense strand of the C-rich sequence overlapping the Sp1 binding site and that, by stabilizing a double stem-loop conformation of this DNA segment, may also play a role in Sp1 regulation of this gene. These studies are the first to describe regulatory and regulated roles for Sp1 in PMA-responsive gene expression and suggest that modulation of Sp1 levels controls thromboxane receptor expression during megakaryocytic differentiation.Thromboxane A 2 is one of the most potent platelet-aggregating and vasoconstricting substances known and is crucial for maintenance of normal hemostasis (1). While considerable attention has been directed toward measuring increased thromboxane production in various disease states including acute coronary ischemic syndromes (2, 3), relatively little is known about how target tissue responses to thromboxane may be modulated. Two intriguing studies have reported increased numbers of platelet thromboxane receptors, with enhanced platelet aggregation, in acute myocardial infarction and unstable angina pectoris (4, 5). The observation that this thromboxane receptor abnormality was reversible after acute myocardial infarction (4) and the discovery of an identical abnormality in patients with unstable angina (the clinical precursor of completed myocardial infarct) (5), strongly sugg...

ABSTRACTa2-Adrenergic receptors comprise a heterogeneous population based on pharmacologic and molecular evidence. We have isolated a cDNA clone (pRNGa2) encoding a rat a2-adrenergic receptor. A rat kidney cDNA library was screened with an oligonucleotide complementary to a highly conserved region found in all biogenic amine receptors described to date. The deduced amino acid sequence displays many features of guanyl nucleotide-binding protein-coupled receptors except it does not have a consensus N-linked glycosylation site near the amino terminus. Membranes prepared from COS cells transfected with pRNGa2 DNA display high affinity and saturable binding to [3H]rauwolscine (Kd = 2 nM).Competition curve data analysis shows that RNGa2 protein binds to a variety of adrenergic drugs with the following rank order of potency: yohimbine 2 chlorpromazine > prazosin 2 clonidine > norepinephrine 2 oxymetazoline. RNGa2 RNA accumulates in both rat kidney and neonatal rat lung (predominant species is 4000 nucleotides). When a cysteine residue (a2-C10), isolated from a human genomic library, encodes an a2A subtype (3), whereas a second clone (a2-C4), isolated from a human kidney cDNA library, encodes a subtype with some of the properties expected of Bylund's a2B subtype (4). For example, the a2-C4 protein has a greater affinity for prazosin than oxymetazoline but is glycosylated following expression of the cDNA in COS cells. Venter and colleagues (5) have further shown that the a2A-adrenergic clone is functional in that Chinese hamster ovary cells transfected with this DNA respond to a2-adrenergic agonists by inhibiting forskolin-stimulated increases in cAMP. Southern blots of human genomic DNA hybridized to a2A-adrenergic receptor DNA showed the existence of three sets of bands, and only two of these were accounted for by known (i.e., a2-C10 and a2-C4) adrenergic receptor genes (3, 4). Thus it appears that there exists a third, closely related gene in the a2-adrenergic subfamily.To obtain the full set of a2-adrenergic clones for our studies on the role of a2-adrenergic receptors in blood pressure control, we screened a rat kidney cDNA library with an oligonucleotide derived from a consensus nucleotide sequence of known biogenic amine receptors. In this report we describe a cDNA encoding a molecule with the ligand binding, tissue distribution, and structural properties expected of an a2B-adrenergic receptor. § METHODS Cloning and Sequence Analysis. Two degenerate oligonucleotides (5'-CTNGAYGTGCTGTKCTGCACSKCSTC-CATCPTGMACCTGTGCG-3' and 5'-CAGSSYGATGPCG-CACAGGT-3'; N = G, A, T, or C; Y = T or C; K = G or T; S = G or C; P = A or G; and M = A or C) were synthesized on a Biosearch 8600 syfnthesizer and purified. The 3' terminal nonamers of these oligonucleotides are complementary; labeled, double-stranded DNA was prepared by the action ofthe Klenow fragment of DNA polymerase I on the annealed oligonucleotide templates in the presence of radiolabeled (32p) deoxynucleoside triphosphates. The resulting 54-residue, 8192-fold degenerat...

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The human platelet thromboxane A2 receptor is a member of the G-protein-coupled superfamily of receptors. Previous pharmacologic studies examining the effects of biochemical reduction, oxidation, or sulfhydryl alkylation on thromboxane receptors have suggested a role for cysteines in determining receptor binding characteristics. To characterize the roles of individual cysteines, we employed site-directed mutagenesis to substitute serines for cysteines at seven positions throughout the human K562 thromboxane receptor and analyzed mutant receptor radioligand ([1S-(1alpha,2beta(5Z),3alpha- (1E,3S),4alpha]-7-[3-(3-hydroxy-4-(p-iodophenoxy)-l-butenyl)-7-oxabicyclo-[2. 2.1]heptane-2-yl]-5-heptenoic acid) binding and calcium signaling. Replacing cysteines in the amino terminus (amino acid position 11), and transmembrane domains two and six (positions 68 and 257) had little effect on thromboxane receptor binding or signaling. Introduction of serines for cysteines in the first (position 105) or the second (position 183) extracellular loop eliminated thromboxane receptor binding, consistent with the existence of a critical disulfide bond between these positions. Mutation of a second cysteine in extracellular loop one (position 102) resulted in a receptor with decreased binding affinity and low binding capacity that transduced only a low amplitude calcium signal, suggesting the involvement of a free sulfhydryl group at this location in receptor-ligand interactions. Finally, mutation of the cysteine at position 223, located in intracellular loop three, resulted in a receptor with normal ligand binding characteristics, but which did not transduce a calcium signal. Some additional amino acid substitutions in this region of the receptor (Cys-223 --> Ala, Thr-221 --> Met) resulted in receptors that had normal binding but transduced low amplitude calcium signals, while other mutations in the same region (His-224 --> Arg and His-227 --> Arg) exhibited normal binding and calcium signaling characteristics. These findings demonstrate that cysteines in extracellular loops one and two contribute to proper ligand binding to thromboxane receptors and show the importance of discrete amino acid sequences in the third intracellular loop, especially cysteine 223, in thromboxane receptor-effector coupling.