David J. Hayzer

Dependence on the Motif YIPP for the Physical Association of Jak2 Kinase with the Intracellular Carboxyl Tail of the Angiotensin II AT1 Receptor

Angiotensin II is the effector molecule of the renin-angiotensin system. Virtually all of its biochemical actions are mediated through a single class of cell-surface receptors called AT1. These receptors contain the structural features of the seven-transmembrane, G-protein-coupled receptor superfamily. Angiotensin II, acting through the AT1 receptor, also stimulates the Jak/STAT pathway by inducing ligand-dependent Jak2 tyrosine phosphorylation and activation. Here, we show that a glutathione S-transferase fusion protein containing the carboxyl-terminal 54 amino acids of the rat AT1A receptor physically binds to Jak2 in an angiotensin II-dependent manner. Deletional analysis, using both in vitro protocols and cell transfection analysis, showed that this association is dependent on the AT1Areceptor motif YIPP (amino acids 319–322). The wild-type AT1A receptor can induce Jak2 tyrosine phosphorylation. In contrast, an AT1A receptor lacking the YIPP motif is unable to induce ligand-dependent phosphorylation of Jak2. Competition experiments with synthetic peptides suggest that each of the YIPP amino acids, including tyrosine 319, is important in Jak2 binding to the AT1A receptor. The binding of the AT1A receptor to the intracellular tyrosine kinase Jak2 supports the concept that the seven-transmembrane superfamily of receptors can physically associate with enzymatically active intracellular proteins, creating a signaling complex mechanistically similar to that observed with growth factor and cytokine receptors.

Cloning and expression of a human endothelin receptor: subtype A.

The polymerase chain reaction, employing degenerate primers specific for the intramembrane domains III and VI of G-coupled receptors, was used to generate partial clones encoding those receptors carried by cultured rat aorta smooth muscle cells. One clone, spanning the intramembrane domains IV-VI of a receptor specific for endothelin-1 (ET-R[A]), was used as a probe to screen a human placental cDNA library. The clone pL4-3, encoding a selective type of human endothelin receptor (ET-R[A]), has an open reading frame encoding a protein 427 amino acids in length, with a relative molecular weight of 48,625 daltons. The sequence analysis suggests the presence of a signal peptide, two potential sites for glycosylation in the N terminal extracellular domain, the seven transmembrane domains typical of G-protein receptors, and several potential sites for phosphorylation in the C terminal cytoplasmic domain. At the amino acid level, the human ET-R(A) shows 91% and 94% identity with the rat and bovine ET-R(A)s, respectively, and 59% similarity with the human ET-R(B). Xenopus laevis oocytes injected with the cloned cDNA express binding sites specific for endothelin-1. Expression of the message in COS 7 cells gave a membrane-bound product to which binding of the [125I]-ET-1 was inhibited by peptide analogues specific for ET-R(A).

The effect of edelfosine on CTP: Cholinephosphate cytidylyltransferase activity in leukemic cell lines

Analogs of ether phospholipids have been shown to have selective anti-neoplastic activity. The compounds are known to inhibit phospholipid biosynthesis. This paper examines the effect of the alkyl-lysophospholipid, edelfosine, on the rate-limiting enzyme, CTP:cholinephosphate cytidylyltransferase, in de novo phosphatidylcholine synthesis in sensitive and resistant leukemic cell lines. Enzyme activity was measured by the incorporation of 14C-phosphocholine into CDP-choline by lysates of HL60 and K562; cells demonstrated inhibition of incorporation of 14C-phosphocholine in HL60 cell lysates but no inhibition in K562 lysates. Partial purification of cytidylyltransferase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting demonstrated similarity between the enzyme isolated from each cell line. Cloning and sequencing of cytidylyltransferase cDNA of HL60 cells was accomplished using a probe encoding the entire protein sequence of the K562 cytidylyltransferase gene. A substitution at nucleotide 751 from A in the HL60 cell cDNA clone to G in the K562 cDNA clone resulted in a change in amino acid number 251 from lysine (positively charged) in the HL60 enzyme to glutamic acid (negatively charged) in the K562 enzyme. This negative charge in the lipid-binding domain of the K562 enzyme may result in a weaker binding of edelfosine and the observed decrease in activity, as evidenced by resistance to edelfosine by K562 cells.

Molecular cloning of a cDNA encoding a novel protein related to the neuronal vesicle protein synaptophysin

The structure of synaptophysin, an integral membrane protein present in synaptic vesicles, is highly conserved. We report the sequence analysis of a clone, HL-5, isolated from a human erythroleukemia cell cDNA library, that is similar to synaptophysin in DNA and amino acid sequence. The predicted protein product derived from this clone is truncated, and the tissue distribution of HL-5 is different from that of synaptophysin. Thus, HL-5 appears to be a member of a previously undescribed family of synaptophysin-like genes.

A rat β-interferon-induced mRNA: sequence characterization

Abstract Polymerase chain reaction amplification of a cDNA derived from rat aortic smooth muscle cells, using sequences from conserved regions of the intramembrane domains of adrenergic receptors as primers, yielded the clone, rat8. This clone possesses a high degree of sequence similarity to a series of human interferon (IFN)-inducible genes. The rat8 sequence is 70% similar to that derived from the human α-IFN-induced gene, 9–27; there is 66% similarity between the deduced amino acid sequences encoded by the rat and the human genes. The rat homologue hybridizes with many bands in Southern analysis of rat DNA, suggesting that it is a member of a large multigene family.

Endothelin A and B Receptors Are Down-Regulated in the Hearts of Hypertensive Rats

Endothelins are vasoactive pep-tides that have been implicated in the development and maintenance of systemic arterial hypertension. The biologic effects of endothelins result from activation of either or both of the two known endothelin receptor subtypes, A and B [ET-R(A) and ET-R(B)], which are present not only in blood vessels but also throughout the cardiovascular and central nervous systems. To investigate the potential role and regulation of myocardial endothelin receptors in hypertension, we examined the expression of ET-R(A) and ET-R(B) receptors in the hearts of normotensive and hypertensive rats. A cDNA probe for the ET-R(A) receptor was obtained by polymerase chain reaction amplification of rat aortic smooth muscle cell mRNA, using degenerate primers specific for intramembrane domains III and VI of G-coupled receptors. Moderate stringency hybridization screening of a rat aortic smooth muscle cell cDNA library yielded a partial clone for the ET-R(B) receptor. These two clones wereusedto examine expression of the ET-R(A) and ET-R(B) receptors in heart, brain, and kidney tissues from Wistar-Kyoto (normotensive), spontaneously hypertensive, salt-hypertensive sensitive, and salt-hypertensive resistant rats by Northern analysis. ET-R(A) and ET-R(B) mRNA were present in the hearts of normal rats. Spontaneously hypertensive rat hearts did not express either ET-R(A) or ET-R(B) mRNA, whereas both salt-hypertensive sensitive and resistant rats fed a high-salt diet expressed both ET-R(A) and ET-R(B) receptor mRNAs. Conversely, in the brain of spontaneously hypertensive rats, mRNAs for both ET-R(A) and ET-R(B) mRNA were present. These observations argue for tissue-specific expression of ET-R(A) and ET-R(B) receptors in spontaneously hypertensive rats, and for differences in endothelin receptor regulation among different models of hypertension in the rat.

Cloning and sequencing of two functional rabbit germ-line immunoglobulin V λ genes

Abstract A recombinant-phage library of rabbit genomic DNA was screened for immunoglobulin VA genes. Two functional genes, Vλ2 and Vλ3, which are separated by 1.6kb were isolated and sequenced. Both are accompanied by signals required for transcription-translation and the recombination with Jλ genes. The two genes, which are 95 % similar in their framework regions, may be the parents of several cDNAs encoding VA regions. Rabbit Vλ-region diversity is likely to be generated by somatic mutation and V-J junction flexibility. Two cDNA clones encoding λ-light chains may have arisen by gene conversion, exchanging the complementarity-determining regions, CDR3s, of the Vλ2 and Vλ3 genes.

Characterization of a cDNA encoding the β-chain of baboon receptor glycoprotein GPIb ☆

A cDNA probe, encoding part of the baboon integrin polypeptide, GPIIb, was used to screen a cDNA library derived from baboon platelet polyadenylated mRNA. One cross-hybridizing clone was found to be 94% similar to a human cDNA encoding the beta-chain of the receptor for von Willebrand factor, a major component of the platelet-subendothelium attachment mechanism.

Strategies for the design of novel thrombolytic and antithrombolytic agents

Recent advances in our understanding of thrombosis and thrombolysis have led to the design of new thrombolytic agents and regimens that may offer improved efficacy. In general, these new approaches specifically target pivotal steps in thrombus formation or lysis. The goal is to reduce adverse side effects (such as bleeding complications) that result from development of a lytic state or that result from a failure to maintain patency (as characterized by rethrombosis). The points in the coagulation cascade that are susceptible to inhibition, as well as the proposed agents for intervention, are discussed in this review.