Protein N-myristoylation refers to the covalent attachment of a myristoyl group (C14:0), via amide linkage, to the NH2-terminal glycine residue of certain cellular and viral proteins. Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes this cotranslational modification. We have developed a system for studying the substrate requirements and biological effects of protein N-myristoylation as well as NMT structure-activit relationships. Expression of the yeast NMT1 gene in Escherchia cofi, a bacterium that has no endogenous NMT activity, results in production of the intact 53-kDa NMT polypeptide as well as a truncated polypeptide derived from proteolytic removal of its NH2-terminal 39 amino acids. Each E. coli-synthesized NMT species has fatty acid and peptide substrate specificities that are indistinguishable from those of NMT recovered from Saccharomyces cerevisiae, suggesting that the NH2-terminal domain of this enzyme is not required for its catalytic activity. By using a dual plasmid system, N-myristoylation of a mammalian protein was reconstituted inE. coliby simultaneous expression ofthe yeastNMT1 gene and a murine cDNA encoding the catalytic (C) subunit of cAMP-dependent protein kinase (PK-A). The fatty acid specificity of N-myristoylation was preserved in this system: [9,10(n)-3H]myristate but not [9,10(n)3H~palmitate was efficiently linked to Gly-1 of the C subunit. [13,14(n)-3HJ10-Propoxydecanoic acid, a heteroatom-containing analog of myristic acid with reduced hydrophobicity but similar chain length, was an effective alternative substrate for NMT that also could be incorporated into the C subunit of PK-A. Such Cotranslational (1) covalent attachment of myristic acid (C14:0) to the NH2-terminal glycine residue of a variety of cellular and viral proteins is, in many instances, required for full expression of their biological activity (reviewed in refs. 2 and 3). Current approaches to understanding the contribution of N-myristoylation to protein structure and function have involved site-directed mutagenesis of the NH2-terminal glycine to prevent acylation or the incorporation of heteroatomcontaining analogs of myristic acid with reduced hydrophobicity into N-myristoylated proteins in vivo (4). For example, abolishing myristoylation of the tyrosine kinase p6Ov-src by deletion of its Gly-1 residue or by Gly-1 -+ Ala substitution revealed (5, 6) that the C14:0 fatty acid is required for the protein's stable association with the plasma membrane (probably through interaction with a high-affinity myristoyl-src receptor; refs. 7 and 8) and its ability to transform cells. Analogous mutagenesis of the Gly-1 residues of the Moloney murine leukemia virus Pr65g (9), the Mason-Pfizer monkey virus Pr7859 (10), and the Pr55n of human immunodeficiency virus I (11) blocks viral replication. X-ray crystallographic studies (12) and site-directed mutagenesis (13) of the N-myristoylated poliovirus capsid protein VP4 have also indicated that myristic acid is involved in protein-protein interactions and in vir...

The human immunodeficiency virus-1 (HIV-1) trans-activator Tat is an attractive target for the development of antiviral drugs because inhibition of Tat would arrest the virus at an early stage. The drug Ro 5-3335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepine-2(H)-one], inhibited gene expression by HIV-1 at the level of transcriptional trans-activation by Tat. The compound did not inhibit the basal activity of the promoter. Both Tat and its target sequence TAR were required for the observed inhibitory activity. Ro 5-3335 reduced the amount of cell-associated viral RNA and antigen in acutely, as well as in chronically infected cells in vitro (median inhibition concentration 0.1 to 1 micromolar). Effective inhibition of viral replication was also observed 24 hours after cells were transfected with infectious recombinant HIV-1 DNA. The compound was active against both HIV-1 and HIV-2 and against 3'-azido-3'-deoxythymidine (AZT)-resistant clinical isolates.

(8,9). In addition to its role as sensor of [Ca 2ϩ ] o , the CaR is also stimulated by aromatic amino acids (10) that, like [Ca 2ϩ ] o , induce striking and lasting CaR-mediated [Ca 2ϩ ] i oscillations (9, 11). However, the patterns of [Ca 2ϩ ] i oscillations induced by these agonists are different. Aromatic amino acid stimulation of the CaR induces repetitive, low frequency [Ca 2ϩ ] i spikes that return to the base-line level, a pattern known as transient oscillations. In contrast, [Ca 2ϩ ] o -elicited CaR activation produces high frequency sinusoidal oscillations upon a raised plateau level of [Ca 2ϩ ] i (9, 11). The amplitude, frequency, and duration of [Ca 2ϩ ] i oscillations are increasingly recognized as encoding important information for a variety of biological processes, and, consequently, there is intense interest in understanding the underlying mechanisms (12).Our previous results produced several lines of evidence indicating that PKCs negatively regulate the frequency of [Ca 2ϩ ] i oscillations induced by activation of the CaR by increases in [Ca 2ϩ ] o (11). We hypothesized that periodic phosphorylation of the CaR by PKCs provides the negative feedback needed to cause [Ca 2ϩ ] o -induced sinusoidal [Ca 2ϩ ] i oscillations. Intriguingly, the transient [Ca 2ϩ ] i oscillations produced by the CaR in response to amino acid stimulation appear to be mediated by a different pathway, but the mechanism(s) involved remained poorly understood.In the present study, we examined whether sinusoidal and transient [Ca 2ϩ ] i oscillations produced by the CaR in response to Ca 2ϩ or L-phenylalanine are mediated by different pathways. Using real time imaging of changes in phosphatidylinositol 4,5-biphosphate hydrolysis and generation of Ins(1,4,5)P 3 in single cells, we found that [Ca 2ϩ ] o -induced CaR activation

Endocytosis of the gastrin releasing peptide receptor (GRP-R) may regulate cellular responses to GRP. We observed endocytosis in transfected epithelial cells by confocal microscopy using cyanine 3-GRP (cyanine 3.18-labeled gastrin releasing peptide) and GRP-R antibodies. At 4 degrees C, cy3-GRP and GRP-R were confined to the plasma membrane. After 5 min at 37 degrees C, ligand and receptor were internalized into early endosomes with fluorescein isothiocyanate-transferrin. After 10 min, cy3-GRP and GRP-R were in perinuclear vesicles, and at 60 min cy3-GRP was in large, central vesicles, while GRP-R was at the cell surface. We quantified surface GRP-R using an antibody to an extracellular epitope and an 125I-labeled secondary antibody. After exposure to GRP, there was a loss and subsequent recovery of surface GRP-R. Recovery was unaffected by cycloheximide, and thus independent of new protein synthesis, but was attenuated by acidotropic agents, and therefore required endosomal acidification. Internalization of 125I-GRP, assessed using an acid wash, was maximal after 10-20 min, and was clathrin-mediated since it was inhibited by hyperosmolar sucrose and phenylarsine oxide. Thus, GRP and its receptor are rapidly internalized into early endosomes and then dissociate in an acidified compartment. GRP is probably degraded whereas the GRP-R recycles.

Understanding the molecular mechanisms of agonistinduced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca 2؉ mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SPinduced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP.

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Protein kinase D (PKD/PKC) immunoprecipitated from COS-7 cells transiently transfected with either a constitutively active mutant of Rho (RhoQ63L) or the Rho-specific guanine nucleotide exchange factor pOnco-Lbc (Lbc) exhibited a marked increase in basal activity. Addition of aluminum fluoride to cells co-transfected with PKD and wild type G␣ 13 also induced PKD activation. Co-transfection of Clostridium botulinum C3 toxin blocked activation of PKD by RhoQ63L, Lbc, or aluminum fluoride-stimulated G␣ 13 . Treatment with the protein kinase C inhibitors GF I or Ro 31-8220 prevented the increase in PKD activity induced by RhoQ63L, Lbc, or aluminum fluoride-stimulated G␣ 13 . PKD activation in response to G␣ 13 signaling was also completely prevented by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Co-expression of C. botulinum C3 toxin and a COOH-terminal fragment of G␣ q that acts in a dominant-negative fashion blocked PKD activation in response to agonist stimulation of bombesin receptor. Expression of the COOH-terminal region of G␣ 13 also attenuated PKD activation in response to bombesin receptor stimulation. Our results show that G␣ 13 contributes to PKD activation through a Rho-and protein kinase C-dependent signaling pathway and indicate that PKD activation is mediated by both G␣ q and G␣ 13 in response to bombesin receptor stimulation.

Cyclooxygenase-2 (COX-2) gene expression is rapidly increased by cytokines, tumor promoters, and growth factors and is markedly enhanced in various cancer cells. Here, we examine the regulation of COX-2 promoter activity by ␣ subunits of heterotrimeric G proteins in NIH 3T3 cells. Using a transient transfection assay with a reporter vector in which the murine COX-2 promoter drives the production of luciferase and expression vectors encoding for ␣ subunits of G-proteins, we show that overexpression of wild type and constitutively active G␣ 13 and G␣ q induced transcription from the COX-2 promoter. The highest level of induced luciferase activity (5.8-fold) occurred in cells expressing the constitutively active G␣ 13 (Q226L). We also show that expression of a constitutively active mutant of Rho (RhoQ63L) also induced transcription from the COX-2 promoter. Co-expression of Clostridium botulinum C3 toxin specifically blocked induction of the COX-2 promoter by either G␣ 13 Q226L or RhoQ63L but did not prevent the activation of this promoter by Ras, Rac, v-src, or forskolin. We conclude that G␣ 13 signals through a Rho-dependent pathway leading to activation of the COX-2 promoter. This pathway is not inhibited by either cytochalasin D, which disrupts actin filament organization, or genistein, a broad spectrum tyrosine kinase inhibitor, indicating a bifurcation of the signaling pathway used by G␣ 13 /Rho to induce COX-2 expression from that used to induce stress fiber formation and tyrosine phosphorylation of focal adhesion proteins.Prostaglandins play a pivotal role in a broad range of physiological and pathological processes including inflammation, pain transmission, maintenance of gastrointestinal integrity, and progression of colorectal cancer (1-3). The rate-limiting enzymes for production of prostaglandins are cyclooxygenases (COX) 1 type 1 and 2 (4 -6). COX-1 is constitutively expressed in nearly all cells, whereas COX-2 expression is induced as an immediate-early gene in response to pro-inflammatory cytokines, tumor promoters, and growth factors (7-10). COX-2 is overexpressed in cancers of the colon, stomach, and breast (11)(12)(13)(14), and chronic inhibition of COX activity has been associated with chemopreventive effects on colon cancer (15). Consequently, the identification of the pathways and regulatory elements that lead to COX-2 expression are the subject of major interest.An increase in the rate of COX-2 gene transcription is mediated by several cis-acting promoter elements that respond to multiple signal transduction pathways (8, 16 -21). Plateletderived growth factor, serum, and v-src promote COX-2 expression through Ras-mediated increases in c-Jun NH2-terminal kinase and extracellular signal-related kinase pathways in NIH 3T3 cells (22)(23)(24). Agonists that signal through cAMP induce COX-2 expression through the cAMP response element binding transcription factor (25). These pathways converge onto a common regulatory region, the ATF/cAMP response element located between Ϫ56 and Ϫ48 of the murine COX...