Enhancement of gene delivery by an analogue of alpha-MSH in a receptor-independent fashion

In order to transfect melanoma specifically by receptor-mediated endocytosis we prepared dioctadecyl aminoglycylspermine (lipospermine)--DNA complexes with [Nle(4),D-Phe(7)]-alpha-MSH(4--10), a pseudo-peptide analogue of alpha-melanocyte stimulating hormone (alpha-MSH) linked to a thiol-reactive phospholipid. With these complexes we obtained an up to 70-fold increase of transfection with B16-F1 melanoma cells. However when B16-G4F, an alpha-MSH receptor negative melanoma cell line was transfected, an up to 700-fold increased transfection efficiency was observed. The peptide hormone analogue was equally efficient when it was only mixed with lipospermine--DNA complexes without covalent coupling. In addition to melanoma cells we also obtained up to 30-fold increased transfection with BN cells (embryonic liver cells). Our data show that an alpha-MSH analogue increased transfection independently of the MSH receptor expression but reaches efficiencies approaching those obtained with peptides derived from viral fusion proteins. The absence of targeting of constructs containing [Nle(4),D-Phe(7)]-alpha-MSH(4-10) can probably be attributed due to the relatively modest number of MSH receptors at the surface of melanoma. We suggest, however, that the peptide hormone analogue used in this study has membrane-active properties and could be of interest as helper agent to enhance non-viral gene delivery presumably by endosomal-destabilizing properties.     

Cloning of Human and Mouse EBI1, a Lymphoid-Specific G-Protein-Coupled Receptor Encoded on Human Chromosome 17q12-q21.2

A lymphoid-specific member of the G-protein-coupled receptor family has been identified by PCR with degenerate oligonucleotides. We have determined that this receptor, also reported as the Epstein-Barr-induced cDNA EBI1, is expressed in normal lymphoid tissues and in several B- and T-lymphocyte cell lines. While the function and the ligand for EBI1 remain unknown, its sequence and gene structure suggest that it is related to the receptors that recognize chemoattractants, such as interleukin-8, RANTES, C5a, and fMet-Leu-Phe. Like the chemoattractant receptors, EBI1 contains intervening sequences near its 5′ end; however, EBI1 is unique in that both of its introns interrupt the coding region of the first extracellular domain. The gene is encoded on human chromosome 17q12-q21.2. None of the other G-protein-coupled receptors has been mapped to this region, but the C-C chemokine family has been mapped to 17q11-q21. The mouse EBI1 cDNA has also been isolated and encodes a protein with 86% identity to the human homolog.     

The receptor for alpha-melanotropin of mouse and human melanoma cells. Application of a potent alpha-melanotropin photoaffinity label

The melanotropin (MSH) receptor of mouse B16-F1 melanoma cells was characterized by photoaffinity cross-linking, using a potent alpha-MSH photolabel, [norleucine4, D-phenylalanine7, 1'-(2-nitro-4-azidophenylsulfenyl)-tryptophan9]-alpha-melanotropin (Naps-MSH). Its monoiodinated form, 125I-Naps-MSH, displayed a approximately 6.5-fold higher biological activity than alpha-MSH. Scatchard analysis of the saturation curves with 125I-Naps-MSH revealed approximately 20,000 receptors/B16-F1 cell and an apparent KD of approximately 0.3 nM. Analysis of the cross-linked MSH receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that a photolabeled band of approximately 45 kDa occurs in B16-F1, B16-F10, and Cloudman S91 mouse melanoma, as well as in human D10 and 205 melanoma but not in non-melanoma cells. The labeled 45-kDa protein had an isoelectric point of 4.5-4.9 as determined by two-dimensional gel electrophoresis. Treatment of the labeled 45-kDa protein of B16-F1 cell membranes by neuraminidase shifted the band to approximately 42 kDa. A similar band of about 42 kDa was also observed after receptor labeling of B16-W4 cells, a cell line with a decreased number of terminal N-linked neuraminyl residues. These results indicate that the labeled 45-kDa glycoprotein contains terminal sialic acid residues, explaining the low pI of this protein, and that it is characteristic for melanoma cells and hence part of the MSH receptor.     

Investigation of the regulation of pigmentation in alpha-melanocyte-stimulating hormone responsive and unresponsive cultured B16 melanoma cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to alpha MSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1 degree) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to alpha MSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro alpha MSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1 degree cells. alpha MSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1 degree cells to produce melanin in response to alpha MSH is not due to a lack of alpha MSH receptors or cAMP response to alpha MSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1 degree and F1 cells.     

Investigation of the Regulation of Pigmentation in α-Melanocyte-Stimulating Hormone Responsive and Unresponsive Cultured B16 Melanoma Cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to αMSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1°) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to αMSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro αMSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1° cells. αMSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1° cells to produce melanin in response to αMSH is not due to a lack of αMSH receptors or cAMP response to αMSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1° and F1 cells.     

Homologous Regulation of the MSH Receptor in Melanoma Cells

Abstract

We have examined the mechanism of homologous regulation of MSH receptor binding and receptor-mediated adenylate cyclase activation in three human and two mouse melanoma cell lines. Pretreatment with α-MSH resulted in a time- and dose-dependent up-regulation of MSH receptors in human D10 and 205 melanoma cells whereas in human HBL and in mouse B16–F1 and Cloudman S91 cells α-MSH induced receptor down-regulation. Up-regulation of receptors was maximal after a 24–h incubation period and an α-MSH concentration of 100 nM (EC₅₀ = 2.4 nM). The increase in α-MSH binding was independent of adenylate cyclase activation and protein synthesis and appeared to be caused by recruitment of spare receptors. The structural requirements of the peptide for triggering this process differed from those found in receptor-binding analyses. Receptor down-regulation was maximal after 12 h and hence more rapid than up-regulation. In B16–F1 cells, 10 nM α-MSH caused the disappearance of 85–90% of the MSH receptors, the EC₅₀ of 0.23 nM lying exactly between that for α-MSH-induced melanogenesis (0.027 nM) and the dissociation constant of receptor binding (1.31 nM). Down-regulation in B16–F1 cells appears to be the consequence of receptor internalization following MSH binding and seems to be initiated during an early step in MSH signalling, preceding the activation of adenylate cyclase and the cAMP signal. Receptor up- and down- regulation were not accompanied by an alteration in affinity to a-MSH, as demonstrated by Scatchard analysis of the binding curves.     

Cloning, structure and expression of a cDNA encoding the human androgen receptor

A cDNA clone has been isolated from a library prepared of mRNA of human breast cancer T47D cells with an oligonucleotide probe homologous to part of the region encoding the DNA-binding domain of steroid receptors. The clone has a size of 1505 bp and sequence analysis revealed an open reading frame of 1356 bp. The deduced amino acid sequence displays two highly conserved regions identified as the putative DNA-binding and hormone binding domains respectively of steroid receptors. Expression of this cDNA clone in COS cells produces a nuclear protein with all the binding characteristics of the human androgen receptor (hAR). The gene encoding the cDNA is assigned to the human X-chromosome. High levels of three hybridizing mRNA species of 11, 8.5 and 4.7 kb respectively are found in the human prostate cancer cell line (LNCaP), which contains elevated levels of hAR. The present data provide evidence that we have isolated a cDNA that encodes a major part of the human androgen receptor.     

Thyrotropin releasing hormone (TRH) selectively binds and activates the melanocortin 1 receptor.

We tested the endogenous tripeptide TRH (thyrotropin releasing hormone) ability to bind to MC (melanocortin) receptor subtypes. We discovered that TRH binds to the human MCI receptor expressed in COS cells and to murine B16 melanoma cells with 5790+/-1010 nM and 6370+/-1260 nM Ki's, respectively. TRH did not bind to the human MC3, MC4 or MC5 receptor subtypes. Moreover, TRH also stimulated cAMP production in murine B16 melanoma cells reaching the same maximum level of cAMP as found for alpha-MSH. However, several analogues of TRH, including TRH-OH, TRH-Gly-NH2 and other analogues, where each of the three amino acid residues in TRH had been exchanged by a related residue, did not bind to any of the MC receptors tested in this study. C(alpha) atoms of molecular models of TRH and the core of a MSH peptide were aligned with r.m.s. of 0.01 A. Moreover, TRH could be docked into a binding pocket of a molecular model of the MC1 receptor at only a little higher energy than a short cyclic MSH peptide. The data indicate a similarity in the mode of TRH and MSH activation of the MCI receptor.     

Extracellular domain of lutropin/choriogonadotropin receptor expressed in transfected cells binds choriogonadotropin with high affinity

The lutropin-choriogonadotropin (LH/CG) receptor is a cell surface receptor comprised of two domains of roughly equivalent size. The amino-terminal half of the receptor is relatively hydrophilic and is located extracellularly, whereas the carboxyl-terminal half of the receptor shares amino acid homology with other receptors that couple to G proteins and is similarly thought to span the plasma membrane seven times, ending with a relatively short carboxyl-terminal tail. In order to test the role of the extracellular domain in binding hormone, we constructed a mutated rat luteal LH/CG receptor cDNA (termed pCLHR-D2), which encodes for only the extracellular domain, and used it to transiently transfect human kidney 293 cells. Here we report that the expressed extracellular domain of the LH/CG receptor is capable of binding human CG with a high affinity, comparable with that of the full-length receptor. Thus, not only is the extracellular domain of the glycoprotein hormone receptors involved in binding hormone, but it alone is capable of conferring high affinity binding. Unexpectedly, it was also found that this truncated receptor is not secreted into the culture media but remains trapped within the cells.     

Anchorage-independent growth of murine melanoma in serum-less media is dependent on insulin or melanocyte-stimulating hormone

α-Melanocyte-stimulating hormone (MSH) is known to stimulate melanogenesis in murine melanoma, particularly in Cloudman S-91 melanoma cells. The effects of MSH and insulin on the proliferation of S91 murine melanoma cells have aroused controversy; in various reports, both hormones have been reported to either stimulate or inhibit murine melanoma growth. In our studies both MSH and insulin stimulated the colony-forming ability and the proliferative capacity of S-91 murine melanoma cells grown in soft agar with either serum-supplemented or serum-less medium. Unless insulin and/or MSH were present, Cloudman S-91 melanoma cells failed to clone in soft agar. The insulin effect was greater than that of MSH, and was more pronounced in serum-less than in serum-supplemented medium. The concurrent treatment of S91 melanoma cells with both MSH and insulin resulted in a greater increase in the total number of colonies formed than caused by treatment with either hormone alone. The combined MSH-insulin stimulation of anchorage-independent growth was specific, since the effect could not be mimicked by epidermal growth factor (EGF), gonadotropin-releasing hormone (GRH), luteinizing hormone (LH), nerve growth factor (NGF) or platelet-derived growth factor (PDGF). Therefore, MSH and insulin may be specific growth factors for murine melanoma cells.     

Desensitization of melanotropin receptors in COS-7 cells

Non-transfected COS-7 cells have been found to possess functional melanotropin receptors on their cell surface. These receptors, and the properties of the melanocyte stimulating hormone (MSH) peptides can be characterized by measuring melanotropin stimulation of cAMP accumulation in the cells. In these cells we studied the ultra-long lasting super agonist [Nle⁴-D-Phe⁷]-α-MSH (NDP-α-MSH), and compared it with the endogenous MSH peptides with respect to potency, maximal activity, duration of action, and rate of desensitization. Surprisingly, NDP- α-MSH did not act as a full agonist in COS-7 cells. In multiple experiments, it could stimulate cAMP accumulation to approximately 50% of the level of α-MSH, β-MSH and adrenocorticotropic hormone (ACTH). The MSH receptor mediating this activity is unknown. The time course of cAMP accumulation, and the duration of receptor activation was also investigated. In contrast to other systems, NDP-α-MSH did not induce prolonged activity, with respect to cAMP accumulation, in COS-7 cells. The MSH receptors present in COS-7 were found to desensitize rapidly subsequent to pretreatment by any of the MSH peptides. As expected for a partial agonist, the activity of NDP-α-MSH desensitized more rapidly than any of the full agonists. Surprisingly, desensitization induced by pretreatment with NDP-α-MSH also occurred more rapidly than desensitization induced by the other MSH analogs.     

Cloning, functional characterization, and expression of a gonadotropin receptor cDNA in the ovary and testis of amago salmon (Oncorhynchus rhodurus).

A gonadotropin receptor was cloned from amago salmon (Oncorhynchus rhodurus) ovarian follicles. This receptor (sGTH-R) belongs to the glycoprotein hormone receptor family with a large extracellular and seven-transmembrane domains. Its sequence homology is highest with mammalian LH receptors. Phylogenetic analysis reveals that sGTH-R is grouped with mammalian and chicken FSH and LH receptors, but not with mammalian TSH receptors. sGTH-R is expressed dominantly in the ovary and testis. Functional characterization examined with transiently transfected mammalian cells revealed increased intracellular cAMP level when exposed to mammalian and fish gonadotropins; the most potent hormone was salmon GTH II. These results indicate that the cloned cDNA encodes a functional amago salmon GTH receptor protein.     

Evidence for increased alpha MSH receptor binding in the F1 variant of B16 melanoma cells grown in dialyzed fetal calf serum

The specific binding of an alpha MSH analogue (Ac-[Nle4, D-Phe7] alpha MSH4-11 NH2) was enhanced in the presence of 10% dialyzed fetal calf serum (FCS) as compared with 10% FCS (nondialyzed) in the F1 variant of B16 melanoma cells. The replenishment of dialyzed serum with adrenocorticotrophic hormone (ACTH) or insulin had no effect on the increased level of alpha MSH receptor binding in these cells. However, 10 nM alpha MSH or 1 microM ACTH under identical conditions significantly decreased the level of alpha MSH binding. Competitive binding studies involving the alpha MSH analogue revealed that the specificity of the receptor was restricted to the complete molecule of alpha MSH, our analogue, beta MSH and ACTH1-24, ACTH4-10, which contains the amino acid sequence responsible for biological activity, showed a very low affinity for the receptor. Furthermore, we observed an interesting phenomenon unique to dialyzed FCS in that once the cells were grown to confluence and melanin was produced, the cells were no longer viable. However, in McCoy's medium, which is deficient in tyrosine, the cells did not produce melanin and remained viable.     

Phosphorylated isomers of L-dopa stimulate MSH binding capacity and responsiveness to MSH in cultured melanoma cells

L-dopa is a key metabolite in the process of melanogenesis. However, it is difficult to use in biological experiments because it is subject to auto-oxidation and relatively insoluble at neutral pH. Dopa phosphates contain phosphate ester linkages at positions 3 and/or 4 of the phenylalanine ring of L-dopa, rendering them highly soluble and stable to auto-oxidation when compared to L-dopa. Dopa phosphates are readily taken up by melanoma cells in culture and converted to L-dopa and inorganic phosphate by cellular phosphatases, making them useful for studying L-dopa effects in vivo. Here we investigated the effects of dopa phosphates on receptors for MSH in cultured melanoma cells. We found that dopa phosphates caused a 3-fold stimulation of MSH binding capacity by the cells which probably occurred through an increase in the number of receptors for MSH with no apparent change in affinity of the receptors. The increased binding capacity for MSH was followed by increased cellular tyrosinase activity and melanogenesis. Thus dopa phosphates and/or L-dopa can act as regulators of the MSH receptor system. The observations suggest a novel mechanism for regulation of hormonal responsiveness: hormonal signal amplification by a metabolite in the target pathway.     

Identification of a gene induced by glucocorticoids in murine T-cells: a potential G protein-coupled receptor.

Using cDNA cloning techniques we previously identified a set of genes induced by glucocorticoids and cAMP in murine T-lymphocytes. We report here the sequence of one of these cDNA clones (clone 4.2), renamed here as glucocorticoid-induced receptor (GIR), which encodes a potential new member of the family of receptors that couple to G-proteins. Several different forms of cDNA for this gene were isolated and shown to correspond to multiple mRNA species in lymphoid cells using an RNase protection assay. The cDNA clone corresponding to the most abundant form of GIR mRNA encodes a precursor protein of 423 amino acids, with a putative signal peptide of 17 amino acids. A hydropathy plot reveals the presence of seven hydrophobic regions, with significant similarities to other G-protein-coupled receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The interleukin-8 receptor is encoded by a neutrophil-specific cDNA clone, F3R.

Interleukin-8 (IL-8) is one of the most potent chemotactic agents for neutrophils and has been implicated as a major mediator of inflammation. The IL-8 receptor is expressed exclusively in neutrophils and belongs to the family of G-protein-coupled receptors. In a recent paper we reported the characterization of a cDNA clone, F3R, isolated from a neutrophil cDNA library and showed that it encodes a G-protein-coupled receptor which is exclusively expressed in neutrophils. We also suggested, based on expression studies in Xenopus oocytes, that the F3R protein product is an isoform of the (fMLP) receptor (Thomas, K. M., Pyun, H. Y., and Navarro, J. (1990) J. Biol. Chem. 265, 20061-20064). In this work, the F3R receptor cDNA is expressed in monkey kidney cells (COS-7) and is shown to encode the IL-8 receptor. F3R cDNA does not encode for a fMLP receptor isoform. We show conclusively that the F3R-transfected COS-7 cells express the IL-8 receptor at a density equivalent to that observed in neutrophils. The pharmacological profile of the F3R-transfected cells is the same as that of neutrophils. The apparent Kd values for binding of 125I-IL-8 to neutrophils and F3R-transfected COS-7 cell membranes were 1.2 and 1.4 nM, respectively. Antipeptide antibodies against a partial sequence of the F3R protein product specifically immunoprecipitate the IL-8 receptor from transfected cells as well as neutrophils. The molecular characterization of the IL-8 receptor should provide the basis for further studies on the identification of the binding domain of this inflammatory receptor.     

Up-regulation of MSH receptors by MSH in Cloudman melanoma cells.

MSH can up-regulate MSH binding capacity of cultured Cloudman melanoma cells in a dose- and time-dependent fashion. Binding is mediated through proteins exhibiting an apparent molecular weight of 50-53kDa, consistent with previous studies implicating them as the principal MSH receptors on Cloudman cells. Pre-incubation of cells with MSH stimulates expression of the receptor proteins both on the plasma membrane surface as well as in internal sites associated with coated vesicles. The effects of MSH are additive with those of UV light, suggesting that UV and MSH might stimulate receptor expression through separate mechanisms.     

Molecular cloning and characterization of a novel human G-protein-coupled receptor, EDG7, for lysophosphatidic acid.

Lysophosphatidic acid (LPA), together with sphingosine 1-phosphate, is a bioactive lipid mediator that acts on G-protein-coupled receptors to evoke multiple cellular responses, including Ca(2+) mobilization, modulation of adenylyl cyclase, and mitogen-activated protein (MAP) kinase activation. In this study, we isolated a human cDNA encoding a novel G-protein-coupled receptor, designated EDG7, and characterized it as a cellular receptor for LPA. The amino acid sequence of the EDG7 protein is 53.7 and 48.8% identical to those of the human functional LPA receptors EDG2 and EDG4, respectively, previously identified. LPA (oleoyl) but not other lysophospholipids induced an increase in the [Ca(2+)](i) of EDG7-overexpressing Sf9 cells. Other LPA receptors, EDG4 but not EDG2, transduced the Ca(2+) response by LPA when expressed in Sf9 cells. LPAs with an unsaturated fatty acid but not with a saturated fatty acid induced an increase in the [Ca(2+)](i) of EDG7-expressing Sf9 cells, whereas LPAs with both saturated and unsaturated fatty acids elicited a Ca(2+) response in Sf9 cells expressing EDG4. In EDG7- or EDG4-expressing Sf9 cells, LPA stimulated forskolin-induced increase in intracellular cAMP levels, which was not observed in EDG2-expressing cells. In PC12 cells, EDG4 but not EDG2 or EDG7 mediated the activation of MAP kinase by LPA. Neither the EDG7- nor EDG4-transduced Ca(2+) response or cAMP accumulation was inhibited by pertussis toxin. In conclusion, the present study demonstrates that EDG7, a new member of the EDG family of G-protein-coupled receptors, is a specific LPA receptor that shows distinct properties from known cloned LPA receptors in ligand specificities, Ca(2+) response, modulation of adenylyl cyclase, and MAP kinase activation.     

AmphiD1/β, a dopamine D1/β-adrenergic receptor from the amphioxus <Emphasis Type="Italic">Branchiostoma floridae</Emphasis>: evolutionary aspects of the catecholaminergic system during development

Catecholamine receptors mediate wide-ranging functions in vertebrates and invertebrates but are largely unknown in invertebrate chordates such as amphioxus. Catecholaminergic cells have been described in amphioxus adults, but few data are known about the transmembrane signal transduction pathways and the expression pattern of related receptors during development. In Branchiostoma floridae, we cloned a full-length cDNA (AmphiD1/β) that corresponds to the dopamine D1/β receptor previously cloned from a related species of amphioxus, Branchiostoma lanceolatum, but no expression studies have been performed for such receptor in amphioxus. In B. floridae, AmphiD1/β encodes a polypeptide with typical G-protein-coupled receptor features, characterized by highest sequence similarity with D1 dopamine and β-adrenergic receptors. The expression of AmphiD1/β mRNA in different regions of the cerebral vesicle corresponds to that of D1-like receptors in vertebrate homologous structures. Furthermore, in situ experiments show that during development, the expression in the nervous system is restricted to cells located anteriorly. A further expression was found in larvae at the level of the endostyle, but it has no counterpart in the predominant expression domains of vertebrate dopamine and/or adrenergic receptor genes. At the same time, we compared the dopaminergic system, consisting of AmphiTH-expressing cells, with the AmphiD1/β expression. In conclusion, the identification of the AmphiD1/β receptor provides further basis for understanding the evolutionary history of the dopaminergic system at the transition from invertebrates and vertebrates.