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- Frändberg, Per-Anders
(author)
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Mutational analysis of melanocortin receptors 1 and 5
- 1998
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Doctoral thesis (other academic/artistic)abstract
- The melanocortin receptor family includes five subtypes, named MC1R to MC5R. Melanocortin receptors are predominantly coupled to second messenger pathway of cyclic AMP through coupling to G-protein. This study is focused on structure-function relationship of MC1R and MC5R. Results demonstrate that (i) mutations D117A and H260A in MC1R decrease the affinity for natural melanocortins (L-isomer) but not for the synthetic analogue NDP-MSH (D-isomer), (ii) reduction of disulphide bonds in MC1R with DTT resulted in decrease in binding of 125I-ACTH in an uniphasic manner and decrease in binding of 125I-NDP-MSH in biphasic manner (iii) single point mutations of four cysteine residues to glycine in extracellular loops of MC1R resulted in a complete loss of ligand binding, (iv) mutant at position C78 in MC1R with wild type ligand binding generated a cAMP signal only in response to α-MSH but not to NDP-MSH, moreover,this single amino acid substitution converted NDP-MSH from being an agonist at wild type receptor to antagonist at this mutant receptor (v) several residues in third intracellular loop of MC1R were identified as necessary for generating a functional response, and a sequence of four amino acids K226-R227-Q228-R229 was found to be essential for coupling of MC1R to G-protein, (vi) a human MC1R variant with a mutation at codon 151 (R151C), cloned from genomic DNA obtained from an individual with red hair and fair skin was found to exhibit wild-type affinities to melanocortins, but was completely nonfunctional, in terms of generating an agonist induced cAMP response (vii) two positions in humanMC5R, Q235 and R272, conserved in all other melanocortin receptors as lysine and cysteine, was demonstrated to raise melanocortin affinities for mutant Q235K and R272C compared to wild-type human MC1R. Thus, this study has identified amino acid residues contributing to ligand interaction and functional activation of melanocortin receptors.
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- Johansson, Tobias, et al.
(author)
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Molecular mechanisms for nanomolar concentrations of neurosteroids at NR1/NR2B receptors
- 2008
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In: Journal of Pharmacology and Experimental Therapeutics. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0022-3565 .- 1521-0103. ; 324:2, s. 759-768
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Journal article (peer-reviewed)abstract
- Neurosteroids are endogenous steroids acting in the central nervous system. They participate in synaptic plasticity, memory and learning, Alzheimer's disease, and certain drug reward. Some mechanisms behind these effects are thought to be nongenomic, e. g., they modulate the function of the N-methyl-D-aspartate (NMDA) receptor complex. In this study, we used a Chinese hamster ovary cell line stably transfected with NMDA receptor constituents NR1/NR2B, to investigate the effects of nanomolar concentrations of the neurosteroids pregnenolone sulfate (PS) and pregnanolone sulfate (3 alpha 5 beta S) on binding of the radioligand [H-3] ifenprodil. Neither of the steroids displaced [H-3] ifenprodil, but both induced a shift in its fit from one to two binding sites. The effects of the neurosteroids were also measured as changes in intracellular calcium ([Ca2+](i)) after glutamate stimulation. Although the steroids did not alter the response to glutamate, they influenced the extent of ifenprodil blockade of the receptor: PS increased and 3 alpha 5 beta S decreased this effect. The coincubation of several NMDA receptor ligands in the assay indicated that PS and 3 alpha 5 beta S act via different binding sites from those for glutamate, glycine, and dithiothreitol. Combining the two steroids revealed that they do not share a common binding site. In conclusion, these results substantiate previous evidence of the allosteric modulatory effect induced by PS and 3 alpha 5 beta S on NMDA receptors at nanomolar concentrations. The neurosteroid-mediated modulation of the receptor is also reflected in an altered glutamate stimulated [Ca2+](i), in response to ifenprodil.
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- Johansson, Tobias, et al.
(author)
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Neurosteroids alter glutamate-induced changes in neurite morphology of NG108-15 cells
- 2007
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In: Journal of Steroid Biochemistry and Molecular Biology. - : Elsevier BV. - 0960-0760 .- 1879-1220. ; 104:3-5, s. 215-219
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Journal article (peer-reviewed)abstract
- Activation of the NMDA receptor leads to increased intracellular Ca2+ levels ([Ca2+]i) which induces outgrowth of and morphologic changes in the neurites of the NG108-15 cell line. This effect can be blocked by antagonists for this glutamate receptor subtype (e.g. ifenprodil or AP5). We have previously shown that nanomolar concentrations of various neurosteroids modulate ifenprodil binding to the NMDA receptor. To investigate whether this interaction affects the functioning of the receptor, we studied the effect of 24 and 48 h of pregnenolone sulphate (PS) or pregnanolone sulphate (3α5βS) on glutamate-stimulated NG108-15 cells. Unexpectedly, the neurosteroids themselves had an inhibitory effect on glutamate-induced changes in neurite patterns. This effect was comparable to that of ifenprodil or AP5. Moreover, the effect of combined treatment with 3α5βS and ifenprodil on neurite morphology indicated a functional interaction between the substances. Interestingly, PS induced cell detachment over time, an effect that was further enhanced by ifenprodil. Cell detachment was also seen after 48 h of treatment with 3α5βS; however, the effect was blocked by ifenprodil and weaker than that of PS. The interaction with the NR2B-selective antagonist ifenprodil indicates that this NMDA receptor subunit may be involved in neurosteroid-induced NG108-15 cell detachment.
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