Genetic construction, expression, and melanoma-selective cytotoxicity of a diphtheria toxin-related alpha-melanocyte-stimulating hormone fusion protein.

Murphy, John R.; Bishai, William R.; Borowski, M; Miyanohara, Atsushi; Boyd, Jessica M.; Nagle, Stanley C.

doi:10.1073/pnas.83.21.8258

Cited by 121 publications

(56 citation statements)

References 41 publications

(14 reference statements)

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“…Besides the pigmenting effect, MSH has also been shown to act as a neurotransmitter in the central nervous system [5], as an endocrine stimulant [6] and as a modulator of immune inflammatory responses [7]. The hormone is considered a potential tool in the diagnosis and therapy of melanoma as it has been used in conjugation with cytotoxin [8] ~r:,~ cytotoxic T-cells [9] for killing melanoma cells by recognizing their MSH receptors. Recently it has been shown that MSH can increase intracellular calcium and activate protein kinase C [10].…”

Section: Introduction Melanocyte Stimulating Hormone (Msh) Is a Stronmentioning

confidence: 99%

Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA

1992

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Melanoeytes and melanoma cells are known to possess receptors for melanoeyte stimulating hormone (MSH). A eDNA clone, designated l ID, has b~.~n isolated from human melanoma cells and encodes a MSH receptor. The cloned eDNA encodes a 317 amino acid protein with transmembrane topography characteristics of a G-protein.coupled receptor, bat it does not show striking similarity to already pablish~ sequences of other G-protein-coupl~ receptors. When 11D eDNA is expressed in COS-7 cells, it binds an Z~l-labelled MSH analogue (NDP-MSH) in a Sl~Cifie manner. The bound ligand could be displaced by melanotropie peptides, a~-MSH, fl.MSH, 7-MSH and ACTH (adrenoeonieotropie hormone), but not by the non-melanotropie ~ptid¢,/3-endorphine. This is the first report of the cloning of a receptor geae of the melanotropin receptor family.

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Section: Introduction Melanocyte Stimulating Hormone (Msh) Is a Stronmentioning

confidence: 99%

Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA

1992

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“…For therapeutic or diagnostic purposes, ligands such as growth factors or the Fv fragments of antibodies are fused to bacterial toxins, (3-lactamase, or alkaline phosphatase, which display useful enzymatic activity when bound to the target cells (1)(2)(3)(4)(5). Also, bifunctional ligands have been created by fusing interleukin 2 (IL2) with IL6, granulocyte/macrophage-colony-stimnulating factor (GM-CSF) with IL3, and IL2 with recombinant antibody fragments (6)(7)(8).…”

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confidence: 99%

A circularly permuted recombinant interleukin 4 toxin with increased activity.

Kreitman

Puri

Pastan

1994

Proc. Natl. Acad. Sci. U.S.A.

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Fusion of ligands such as growth factors to other proteins often dramatically reduces the affinity of the ligand for its receptor. With recombinant DNA techniques, the attachment point between the two proteins has until now been restricted to either the amino or the carboxyl terminus of the d. However, binding may be greatly compromised if both ends are close to the site at which the ligand binds to its receptor. To construct a single-chain growth factor fusion protein with the connection at a new site on the growth factor, we constructed a DNA fria ent encoding circularly permuted interleukin 4 (H4), termed 114(38-37 In many cases, fusion of one protein to another impairs the activity of one or both proteins. This can occur if one or both proteins require one or both termini free for optimal activity. Alternatively, connection of one protein to another can impair the ability ofeither protein to properly fold. It has been reported that transforming growth factor a, IL2, IL3, IL4, IL6, GM-CSF, and insulin-like growth factor I all bind with 20-to 250-fold reduced affinity after fusion to another protein (7,(13)(14)(15)(16). Previously, the options available for improving the function of a fusion protein were limited to switching the order of the two proteins, attaching linkers in between the two proteins, or mutating one of the proteins to decrease junctional effects (13,17).We have previously reported on the properties of chimeric toxins in which IL4 was fused to recombinant forms of Pseudomonas exotoxin (PE). The fusion proteins bind to the IL4 receptor (IL4R) with only "l4% of the affinity of native IL4 (14). Several studies have indicated that the carboxyl terminus of IL4 is important for binding (18,19); therefore, it is likely that the large toxin molecule attached to the carboxyl terminus blocks the binding of 1L4 to the IL4R. We therefore developed a strategy for fusing the two proteins in which the toxin is fused to the new carboxyl terminus of circularly permuted forms of IL4. A circular permuted protein is a mutant protein in which the termini have been fused and new termini created elsewhere in the molecule. Several circularly permuted proteins have been made but none of these have been fused to other proteins (20). We reasoned that circularly permuted IL4 (CP-IL4) could be fused to a toxin so that thejunction would be in an entirely new location and allow the IL4 to bind in a more native fashion. MATERIALS AND METHODSPlasmid Construction. pRKL4EL encodes human IL4 followed by the amino acids ELKA, contains gag-ctc-gaa-gcttga (nucleotide sequences are shown by lowercase letters to avoid confusion with single-letter amino acid symbols) at the end of the coding sequence, and was derived by PCR amplification of pWDMH4 (14) and ligation into the vector pVCDT1-anti-Tac(Fv) (21). pRKL4 encodes the 129 amino acid 14 protein purified from Escherichia coli (22) and was derived from PCR amplification of pRKLAEL and ligation into the vector pVCDT1-anti-Tac(Fv). DNA encoding the toxin used for fusion to CP...

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“…This dream was realized almost a hundred years later by Murphy et al (1986) with the design and synthesis of DT based fusion protein toxins that were targeted toward specific eukaryotic cell receptors. Substitution of the native R domain with a surrogate ligand results in the formation of a fusion protein toxin construct that targets cells expressing the appropriate cell surface receptor.…”

Section: Receptor Bindingmentioning

confidence: 99%