The cellular tropisms of eukaryotic viruses are shaped by their need for entry receptors and intracellular transcription factors. Here we show that viral tropisms can also be regulated by tissue-specific microRNAs (miRNAs). Target sequences complementary to muscle-specific miRNAs were inserted into the 3' untranslated region (UTR) of an oncolytic picornavirus that causes lethal myositis in tumor-bearing mice. The recombinant virus still propagated in subcutaneous tumors, causing total regression and sustained viremia, but could not replicate in cells expressing complementary miRNAs and therefore did not cause myositis. This altered tropism was not due to insertional attenuation, as a control virus containing a 3' UTR insert with a disrupted miRNA target sequence fully retained its lethal myotropism. Tissue-specific destabilization of viral genomes by miRNA target insertion provides a potentially versatile new mechanism for controlling the tropism of replicating viruses for therapy and may serve as a new modality for attenuating viruses for vaccine purposes.

Mechanisms of ligand binding and activation of G protein-coupled receptors are particularly important, due to their ubiquitous expression and potential as drug targets. Molecular interactions between ligands and these receptors are best defined for small molecule ligands that bind within the transmembrane helices. Extracellular domains seem to be more important for peptide ligands, based largely on effects of receptor mutagenesis, where interference with binding or activity can reflect allosteric as well as direct effects. We now take the more direct approach of photoaffinity labeling the active site of the cholecystokinin (CCK) receptor, using a photolabile analogue of CCK having a blocked amino terminus. Guanine nucleotide-binding protein (G protein) 1 -coupled receptors are the largest group of plasma membrane receptors, representing a superfamily with a remarkable diversity of activating ligands. Our best understanding of the molecular basis for ligand binding to members of this superfamily is the binding of the chromophore to rhodopsin and the binding of biogenic amines to adrenergic receptors. These insights come from complementary studies of receptor mutagenesis, photoaffinity labeling, and reciprocal chemical modification of ligand and receptor (1-6). All available data focus the relevant interactions to sites at the core of the coalescence of transmembrane helices, in the outer third of the bilayer. Even with this extensive information, the constrained nature of the ligands, and the relatively confined space for ligand docking, the debate continues regarding the specific siting of the agonist ligands in some of these receptor systems (7,8).Understanding the interactions between peptide ligands and their G protein-coupled receptors represents an even greater challenge. By first principles, these ligands are quite flexible and can achieve many conformations. Whereas some peptides appear to have some preferred conformation in solution (9), there is little information regarding how such structures relate to the receptor-bound states of these ligands. Most of our insights into binding domains for peptide ligands have come from receptor mutagenesis studies, which have focused attention on receptor domains predicted to be outside the membrane (7,8). Given the extended size of the pharmacophoric domains and the solubilities of the peptide ligands, these regions of interaction seem plausible. We know, however, that receptor mutagenesis can modify receptor function nonspecifically, interfering with biosynthetic processing or trafficking or having an allosteric effect, rather than necessarily directly interfering with a site of ligand-receptor interaction. For a very limited number of peptide receptors in this family, direct sites of contact have been recently described using photoaffinity labeling approaches (10 -13).Cholecystokinin (CCK) is a peptide hormone and neurotransmitter that has a wide spectrum of physiologic actions (14). These relate largely to control of nutrient assimilation, through regulation of...

]secretin-27 probe was a fully efficacious agonist, with a potency to stimulate cAMP accumulation by Chinese hamster ovary SecR cells similar to that of natural secretin (EC 50 ‫؍‬ 68 ؎ 22 pM analogue and 95 ؎ 25 pM secretin). It bound specifically and with high affinity (K i ‫؍‬ 5.0 ؎ 1.1 nM) and covalently labeled the M r ‫؍‬ 57,000-62,000 secretin receptor. Cyanogen bromide cleavage of the receptor yielded a major labeled fragment of apparent M r ‫؍‬ 19,000 that shifted to M r ‫؍‬ 9,000 after deglycosylation. This was most consistent with either of two glycosylated domains within the amino-terminal tail of the receptor. Immunoprecipitation with antibody directed to epitope tags incorporated into each of the candidate domains established that the fragment at the amino terminus of the receptor was the site of labeling. This was further localized to the amino-terminal 30 residues of the receptor by additional proteolysis of this fragment with endoproteinase Lys-C. This provides the first direct demonstration of a contact between a secretin-like agonist and its receptor and will contribute a useful constraint to the modeling of this interaction.The secretin receptor is prototypic of a recently recognized family (Class II) of guanine nucleotide-binding protein (G protein) 1 -coupled receptors (1). Members of this family are believed to have the seven-transmembrane segment topology typical of the superfamily, but they share Ͻ12% homology with the extensively studied Class I receptors in the rhodopsin/␤-adrenergic receptor family, and they lack the signature sequences of this family (2, 3). Secretin family receptors have long aminoterminal domains incorporating six highly conserved Cys residues, believed to contribute to disulfide bonds that help define the family (3, 4). Indeed, this complex domain has been suggested to play a key role in agonist binding, as suggested by receptor mutagenesis studies (5-9). Other extracellular loop domains have also been implicated in complementary roles for agonist binding and receptor activation (4,5,7,10). Natural ligands for this family of receptors are all peptides longer than 27 residues, with structure-activity series suggesting the presence of diffuse pharmacophoric domains (3). Although this large diffuse pharmacophore nicely complements the multiple domains predicted to be outside the membrane bilayer, there is no working model to predict how the two molecules might interact.In this work, we attempt to establish an initial constraint that will contribute to the development of a model for the interaction of secretin with its receptor. We do this through photoaffinity labeling. This has the theoretical advantage of directly probing the domain adjacent to the photolabile residue within the probe after it binds to the receptor. Using this approach, we have successfully identified two binding contacts between photolabile analogues of cholecystokinin and its receptor (11,12).In this work, we have developed an analogue of secretin that incorporates a site for radioiodination an...

Abstract. Receptor molecules play a major role in the desensitization of agonist-stimulated cellular responses. For G protein-coupled receptors, rapid desensitization occurs via receptor phosphorylation, sequestration, and internalization, yet the cellular compartments in which these events occur and their interrelationships are unclear. In this work, we focus on the cholecystokinin (CCK) receptor, which has been well characterized with respect to phosphorylation. We have used novel fluorescent and electron-dense CCK receptor ligands and an antibody to probe receptor localization in a CCK receptor-bearing CHO cell line. In the unstimulated state, receptors were diffusely distributed over the plasmalemma. Agonist occupation stimulated endocytosis via both clathrin-dependent and independent pathways. The former was predominant, leading to endosomal and lysosomal compartments, as well as recycling to the plasmalemma. The clathrin-independent processes led to a smooth vesicular compartment adjacent to the plasmalemma resembling caveolae, which did not transport ligand deeper within the cell. Potassium depletion largely eliminated clathrin-dependent endocytosis, while not interfering with agoniststimulated receptor movement into subplasmalemmal smooth vesicle compartments. These cellular endocytic events can be related to the established cycle of CCK receptor phosphorylation and dephosphorylation, which we have previously described (Klueppelberg,

No abstract

Secretin and vasoactive intestinal polypeptide (VIP) receptors are closely related G protein-coupled receptors in a recently described family possessing a large amino-terminal ectodomain. We postulated that this domain might be critical for agonist recognition and therefore constructed a series of six chimeric receptors, exchanging the amino terminus, the first extracellular loop, or both in secretin and VIP receptors. Constructs were expressed in COS cells and characterized by cAMP generation and binding of both secretin and VIP radio-ligands. Wild type receptors demonstrated high affinity binding of respective ligands (IC50 values (in nM): at the secretin receptor: 2.2 for secretin, > 1000 for VIP; at the VIP receptor: 2.2 for VIP, > 1000 for secretin) and appropriately sensitive and selective biological responses (EC50 values (in nM): at the secretin receptor: 1.5 for secretin, 127 for VIP; at the VIP receptor: 1.0 for VIP, 273 for secretin). Replacement of the secretin receptor amino terminus with that of the VIP receptor resulted in biological responsiveness typical of the VIP receptor (EC50 = 120 nM for secretin, 1.7 nM for VIP). The converse was not true, with this domain of the secretin receptor not able to provide the same response when incorporated into the VIP receptor (EC50 = 50 nM for VIP, 30 nM for secretin). The addition of both the first loop and the amino terminus of the secretin receptor was effective in yielding a secretin receptor-like response (EC50 = 2.0 nM for secretin, 47 nM for VIP). All chimeric constructs expressing selectivity for secretin-stimulated activity bound this hormone with high affinity (IC50 = 0.2-2.2 nM); however, there was divergence between VIP binding and biological activity. Thus, the amino terminus of secretin and VIP receptors plays a key role in agonist recognition and responsiveness, with the first loop playing a critical complementary role for the secretin receptor.

No abstract

Oncolytic viruses (OV) are promising treatments for cancer, with several currently undergoing testing in randomised clinical trials. Measles virus (MV) has not yet been tested in models of human melanoma. This study demonstrates the efficacy of MV against human melanoma. It is increasingly recognised that an essential component of therapy with OV is the recruitment of host anti-tumour immune responses, both innate and adaptive. MV-mediated melanoma cell death is an inflammatory process, causing the release of inflammatory cytokines including type-1 interferons and the potent danger signal HMGB1. Here, using human in vitro models, we demonstrate that MV enhances innate antitumour activity, and that MV-mediated melanoma cell death is capable of stimulating a melanoma-specific adaptive immune response.