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J E Wikberg

Publications and source records attributed to J E Wikberg.

At least 73 records · Page 4Linked to original sources
PubMed1997

Autoradiographic studies of central alpha 2A- and alpha 2C-adrenoceptors in the rat using [3H]MK912 and subtype-selective drugs.

In the present study we examined the distribution of alpha 2A- and alpha 2C-adrenoceptors in tissue slices from the rat cervical spinal cord and from brain slices collected at the level of the striatum. To differentiate between alpha 2A- and alpha 2C-adrenoceptors, the slices were incubated with [3H]MK912 in the presence of graded concentrations of the alpha 2A-selective drug, BRL44408, or the alpha 2C-selective drug, spiroxatrine. Computer analysis of the autoradiograms indicated that 0.4 nM [3H]MK912 plus 185 nM BRL44408 selectively labeled alpha 2C-adrenoceptors, while 0.4 nM [3H]MK912 plus 220 nM spiroxatrine selectively labeled alpha 2A-adrenoceptors. Using this approach, alpha 2C-adrenoceptors were detected in the striatum, while alpha 2A-adrenoceptors predominated in the cortical layers 1-4, the spinal cord distal dorsal horn, the septum and the endopiriform nucleus.

Adrenergic alpha-Antagonists↗
PubMed1997

Deletions of the N-terminal regions of the human melanocortin receptors.

The non-homologous N-terminal regions of four human melanocortin (MC) receptors were truncated in order to investigate their putative participation in ligand binding. Eleven constructs were made, where different numbers of residues from the N terminus were deleted. These constructs were used for transient expression experiments in COS cells and analysed by ligand binding. The results show that 27, 25, 28, and 20 amino acids could be deleted from the N terminus of the human MC1, MC3, MC4 and MC5 receptors, respectively, including all potential N-terminal glycosylation sites in the MC1 and the MC4 receptors, without affecting ligand binding or expression levels. The results indicate that the N-terminal regions of the human MC1, MC3, MC4 and MC5 receptors, do not play an important role for the ligand binding properties of these receptors.

Amino Acid Sequence↗
PubMed1997

Characterisation of D117A and H260A mutations in the melanocortin 1 receptor.

Recent site directed mutagenesis studies on the melanocortin 1 (MC1) receptor have indicated the importance of D117 and H260 amino acid residues for the binding of alpha-MSH (melanocyte stimulating hormone). Here, we report the testing of 12 cyclic and linear MSH peptides on the D117A and H260A mutant receptors. Moreover, we constructed a double mutant which displayed a major loss in affinity for [Nle4, D-Phe7]alpha-MSH. Our new data of His6 and Phe7 substituted MSH peptides are compared with previous results and the hypothesis of putative interactions of D117 and H260 with single amino acids in the MSH peptide. Our conclusions are that the D117A and the H260A mutations may cause conformational changes in the receptor which can not be linked to any specific amino acid in the MSH-peptides.

Animals↗
PubMed1997

Binding of cyclic and linear MSH core peptides to the melanocortin receptor subtypes.

We report here the binding of 5-, 6- and 7-amino-acid-long linear and cyclic core peptides of MSH (melanocyte-stimulating hormone) to cells transiently expressing the human melanocortin MC1, MC3, MC4 and MC5 receptors. The results show that, in contrast to the natural peptides, the core peptides did not differentiate between the melanocortin MC3 and MC4 receptors. All tested cyclic peptides had much lower affinities than their corresponding linear homologues. Interestingly, the relative loss of binding due to the cyclisation did not change as the ring size decreased. Therefore, decreasing the ring size does not seem to force the peptide into a more unfavourable conformation.

Adrenocorticotropic Hormone↗
PubMed1997

Postnatal expression of melanocortin-3 receptor in rat diencephalon and mesencephalon.

In situ hybridization was applied to examine the postnatal expression of melanocortin-3 (MC-3) receptor mRNA in the rat brain. Very weak and limited signals were seen in the hypothalamus on postnatal day 0 (P0) and in the dorsal lateral thalamus on P4. A marked increase was noted in several regions of the diencephalon and mesencephalon on P7. The highest levels were reached on P21, which was the time when an adult-like pattern was established. On P21, intense signals were seen in the ventromedial nucleus and the arcuate nucleus of the tuberal hypothalamus, the habenular nucleus of the epithalamus and the ventral tegmental area. [125I] Nle4, D-Phe7-alpha-MSH showed overlapping, but wider labelling of melanocortin receptors, that followed a similar developmental course. alpha-MSH-like immunoreactivity was seen widely in the forebrain and midbrain from P14. In contrast to the staining of alpha-MSH in neurons and their process, gamma 2-MSH-like immunoreactivity was detected strongly in the blood vessels. The neuronal localization of MC-3 receptor mRNA suggests that this receptor may mediate the neurotropic actions of melanocortin peptides in the developing brain.

Animals↗
PubMed1997

Characterization of the binding of MSH-B, HB-228, GHRP-6 and 153N-6 to the human melanocortin receptor subtypes.

We determined the binding affinities of the MSH analogues MSH-B, HP-228 and 153N-6 and of the enkephalin analogue GHRP-6 on a single eukaryotic cell line transiently expressing the human MC1, MC3, MC4 and MC5 receptors. Moreover, we tested the binding and cAMP response of MSH-B in comparison with alpha-MSH on murine B16 melanoma cells. Our results indicate that MSH-B has a potency similar to that of alpha-MSH and that these two peptides induce similar cAMP responses in murine B16 melanoma cells. HP-228 has its highest affinity for the MC1 receptor. For the other receptors, it has slightly higher affinity for the MC5 receptor than for the MC3 and MC4 receptors. 153N-6 was found to be selective for the MC1 receptor. GHRP-6 was found to bind to the MC1 and the MC5 receptors despite its low structural homology with alpha-MSH. [D-Lys3]GHRP-6 bound to all the four MC receptors with similar affinities. The structurally related Met-enkephalin and the functionally related GHRH, as well as LHRH and somatostatin-14 did not bind to these MC receptors. The low affinity of the GH-releasing/enkephalin peptides may indicate that they do not interact with the MC receptors at pharmacologically relevant concentrations.

Animals↗
PubMed1997

Selectivity of cyclic [D-Nal7] and [D-Phe7] substituted MSH analogues for the melanocortin receptor subtypes.

The binding of the 2 cyclic lactam MSH (4-10) analogues (MTII, SHU9119), and 5 cyclic [Cys4, Cys10] alpha-MSH analogues were tested on cells transiently expressing the human MC1, MC3, MC4 and MC5 receptors. The results indicate a differential importance of the C-terminal (Lys-Pro-Val) and N-terminal (Ser-Tyr-Ser) of cyclic [Cys4, Cys10] alpha-MSH analogues in binding to the MC receptor subtypes. Substitution of D-Phe7 by D-Nal(2')7 in both the cyclic lactam MSH (4-10) and the cyclic disulphide MSH (4-10) analogues resulted in a shift in favour of selectivity for the MC4 receptor; the disulphide analogue, [Cys4, D-Nal(2')7 Cys10] alpha-MSH (4-10) (HS9510), showing the highest selectivity for the MC4 receptor among all the substances tested. However, the cyclic lactams displayed an over all higher affinity for the MC receptors, than any of the cyclic disulphide MSH (4-10) analogues.

Animals↗
PubMed1997

Selectivity of [Phe-I7], [Ala6], and [D-Ala4,Gln5,Tyr6] substituted ACTH(4-10) analogues for the melanocortin receptors.

We tested [Ala6]ACTH(4-10) and [Phe-I7]ACTH(4-10)(putative MC receptor antagonists), [D-Ala4,Gln5,Tyr6]ACTH(4-10)(BIM 22015), and ACTH (4-10) with radioligand binding using transiently expressed human MC1, MC3, MC4, and MC3 receptors. [Phe-I7]ACTH(4-10) had higher affinity for the MC3, MC4, and MC3 receptors but lower for the MC1 compared to ACTH(4-10). [Ala6]ACTH(4-10) did not bind the MC1 receptor but had highest affinity for the MC4 receptor. The data indicate that the His6 has a specially important role in binding to the MC1 receptor. The BIM 22015 did not bind to these MC receptor subtypes, which indicates that the neurotrophic and myotrophic properties that are attributed to this peptide are mediated by some other receptor.

Adrenocorticotropic Hormone↗
PubMed1997

A series of novel imidazoline I2-receptor selective Schiff bases of 1-(benzylidenamino)-3,3-dimethylguanidine.

A series of 12 Schiff bases of 1-(benyzylidenamino)-3,3-dimethylguanidine were evaluated for their affinities at imidazoline I2-receptors and alpha 2-adrenoceptors in the guinea pig cerebral cortex by using a 2-curve assay with [3H]idazoxan as ligand and the simultaneous computer analysis of (-)adrenaline masked and non-masked competition curves. All of the dimethylguanidines were selective for I2-receptors with the selectivity ranging 6-110-fold, and the most selective being WAP8 (2-bromobenzylidenamino-3,3-dimethylguanidine) closely followed by FLA151 (2-chlorobenzylidenamino-3, 3-dimethylguanidine). Some of the dimethylguanidines were also tested for affinity at bovine kidney cortex I2-receptors and guinea pig alpha 1-adrenoceptors. These substances were found to have similar affinities for bovine kidney I2-receptors as for guinea pig cerebral cortex I2-receptors, but their affinities for the alpha 1-adrenoceptors were considerably lower.

Animals↗
PubMed1997

Modeling of the three-dimensional structure of the human melanocortin 1 receptor, using an automated method and docking of a rigid cyclic melanocyte-stimulating hormone core peptide.

A model is presented of the melanocortin 1 receptor (MC1R), constructed by use of an unbiased, objective method. The model is created directly from data derived from multiple sequence analysis, a low-resolution EM-projection map of rhodopsin, and the approximate membrane thickness. The model agrees well with available data concerning natural mutations of MC1Rs occurring in different species. A model is also presented of the most rigid ligand for this receptor, the cyclic pentapeptide cHFRWG, shown docked in the receptor model. The receptor-ligand complex model agrees well with available experimental data. The ligand is located between transmembrane region 1 (TM1), TM2, TM3, TM6, and TM7 of the receptor. Multiple interactions occur between ligand and receptor, including interactions with Leu-48 (TM1), Ser-52 (TM1), Glu-55 (TM1), Asn-91 (TM2), Glu-94 (TM2), Thr-95 (TM2) Ile-98 (TM2), Asp-121 (TM3), Thr-124 (TM3), Phe-257 (TM6), Phe-283 (TM7), Asn-290 (TM7), and Asp-294 (TM7) of the receptor.

Amino Acid Sequence↗
PubMed1997

Quantification of alpha2A and alpha2C adrenoceptors in the rat striatum and in different regions of the spinal cord.

The alpha2C-adrenoceptor preferring radioligand [3H]-MK912 was used for labelling alpha2A- and alpha2C-adrenoceptors in the rat striatum, in the cervical, thoracic and lumbar parts of the spinal cord, and in the dorsal and ventral halves of the spinal cord. In addition, guanfacine was used as a tool to delineate the alpha2A- and alpha2C-adrenoceptors. In the striatum the sites were 72% alpha2A- and 28% alpha2C-adrenoceptors, while in all regions of the spinal cord the proportions of the sites were about 96% alpha2A- and 4% alpha2C-adrenoceptors. A multi-curve experimental design and computer analysis was used in order to enable the accurate quantification of the alpha2A- and alpha2C-adrenoceptors in the striatum and spinal cord.

Adrenergic alpha-Agonists↗
PubMed1997

The melanocortin 1, 3, 4 or 5 receptors do not have a binding epitope for ACTH beyond the sequence of alpha-MSH.

ACTH(1-39), and several shorter N- and/or C-terminally truncated fragments of ACTH, with and without N-terminal acetylation and/or C-terminal amidation, were tested for binding on a single eukaryotic cell line transiently and independently expressing the melanocortin MC1, MC3, MC4 and MC5 receptors. The results show that none of these MC receptors has specific binding epitopes for the ACTH peptides beyond the amino acid sequence of alpha-MSH, when tested for their ability to compete with 125I-labelled [Nle4,D-Phe7]alpha-MSH and ACTH. The MC3 receptor favours the natural desacetylated N-terminal end of the ACTH peptides, and it has generally more than 10-fold higher affinity for the ACTH peptides than the MC4 receptor. Considering earlier anatomical localisation data, together with the present data, we suggest that the MC3 receptor is the most likely candidate of the MC receptors to mediate the short-loop negative feedback release of corticotrophin-releasing factor (CRF) caused by ACTH/MSH peptides.

Adrenocorticotropic Hormone↗
PubMed1996

Evidence indicating that the TM4, EL2, and TM5 of the melanocortin 3 receptor Do not participate in ligand binding.

The TM4, EL2 and TM5 show low amino acid homology within the MC receptor family. Three mutants of the human MC3 receptor were created in order to investigate the participation of these regions in ligand binding. The TM4, EL2 and TM5 were separately changed by multiple mutagenesis so that their amino acid sequences became identical with the human MC1 receptor. The mutants were expressed in COS cells and they bound peptide ligands in the same fashion as the wild type MC3 receptor clone. Our results indicate that the amino acids that were mutated in the MC3 receptor do not affect the binding of MSH peptides. The data provide further evidence, that the mutated regions may not participate at all in ligand binding, as indicated by modelling experiments and homology comparison.

Animals↗
PubMed1996

Alternative translation initiation codon for the human melanocortin MC3 receptor does not affect the ligand binding.

The genomic DNA for the human melanocortin MC3 receptor indicates an unusually long N-terminus. Two possible translation initiation sites, the one originally proposed and one alternate 111 bp downstream, were mutated. For a third mutant the DNA between these initiation sites was deleted. All mutants were expressed in COS (CV-1 Origin, SV40) cells in the same level, and they bound peptide hormones in the same fashion, as did the wild type clone. The data obtained indicate that both sites can function as the sole translation initiation sites of the human clone and that the proposed N-terminus of the human melanocortin MC3 receptor is not important for the ligand binding of the receptor.

Binding, Competitive↗
PubMed1996

Expression of functional melanocortin 1 receptors in insect cells.

We expressed epitope-tagged human melanocortin 1 receptor (MC1R) in insect cells using two different recombinant baculovirus constructs; one of which encoded MC1R with an N-terminal Flag epitope and a C-terminal polyHis tag, while the other encoded the MC1R with a C-terminal Myc tag. The constructs were used to infect Sf9 insect cells. For both constructs, immunoblotting with tag-specific antibodies demonstrated the presence of the receptor in the infected cells. The infected Sf9 cells were characterized by radioligand binding using [125I][Nle4,D-Phe7]alpha-MSH. Both saturation and competition analysis, using alpha-, beta-, and gamma 1-MSH on the tagged MC1R expressed in the insect cells, gave binding constants and potency orders that were undistinguishable from those obtained on MC1R expressed in COS cells. The expression level obtained (in the order of pmoles of binding sites per mg of protein) will now facilitate attempts to purify the receptor. This is the first report that demonstrates a functional expression of recombinant melanocortin receptor in nonmammalian cells.

Amino Acid Sequence↗
PubMed1996

Immunological localisation of melanocortin 1 receptor on the cell surface of WM266-4 human melanoma cells.

The localisation of melanocortin 1 receptor (MC1R) in WM266-4 human melanoma cells was investigated by applying an antipeptide antiserum specific for the cloned human MC1R (MSH receptor). In enzyme-linked immunosorbent assay (ELISA), the immunoreactivity was detected in the membrane fraction of WM266-4 cells. The ELISA reactivity could be inhibited by an antiserum pre-absorbed with its specific synthetic peptide. In immunocytochemistry, the specific immunoreactivity was demonstrated on the surface of the cells by using either biotin-avidin immunoalkaline phosphatase- or TRITC-staining method. These results indicate that the MC1R is prominently present on the plasma membrane of WM266-4 human melanoma cells.

Affinity Labels↗