Recombinant MT2 Research Articles

Melatonin inhibits insulin secretion in rat insulinoma β-cells (INS-1) heterologously expressing the human melatonin receptor isoform MT2

Melatonin exerts some of its effects via G-protein-coupled membrane receptors. Two membrane receptor isoforms, MT1 and MT2, have been described. The MT1 receptor is known to inhibit second messenger cyclic adenosine monophosphate (cAMP) signaling through receptor-coupling to inhibitory G-proteins (G(i) ). Much less is known about the MT2 receptor, but it has also been implicated in signaling via G(i) -proteins. In rat pancreatic β-cells, it has recently been reported that the MT2 receptor plays an inhibitory role in the cyclic guanosine monophosphate (cGMP) pathway. This study addresses the signaling features of the constitutively expressed human recombinant MT2 receptor (hMT2) and its impact on insulin secretion, using a rat insulinoma β-cell line (INS-1). On the basis of a specific radioimmunoassay, insulin secretion was found to be more strongly reduced in the clones expressing hMT2 than in INS-1 controls, when incubated with 1 or 100 nm melatonin. Similarly, cAMP and cGMP levels, measured by specific enzyme-linked immunosorbent assays (ELISAs), were reduced to a greater extent in hMT2 clones after melatonin treatment. In hMT2-expressing cells, the inhibitory effect of melatonin on insulin secretion was blocked by pretreatment with pertussis toxin, demonstrating the coupling of the hMT2 to G(i) -proteins. These results indicate that functional hMT2 expression leads to the inhibition of cyclic nucleotide signaling and a reduction in insulin release. Because genetic variants of the hMT2 receptor are considered to be risk factors in the development of type 2 diabetes, our results are potentially significant in explaining and preventing the pathogenesis of this disease.

Cadmium binding studies to the earthworm Lumbricus rubellus metallothionein by electrospray mass spectrometry and circular dichroism spectroscopy

The earthworm Lumbricus rubellus has been found to inhabit cadmium-rich soils and accumulate cadmium within its tissues. Two metallothionein (MT) isoforms (1 and 2) have been identified and cloned from L. rubellus. In this study, we address the metalation status, metal coordination, and structure of recombinant MT-2 from L. rubellus using electrospray ionization mass spectrometry (ESI-MS), UV absorption, and circular dichroism (CD) spectroscopy. This is the first study to show the detailed mass and CD spectral properties for the important cadmium-containing earthworm MT. We report that the 20-cysteine L. rubellus MT-2 binds seven Cd 2+ ions. UV absorption and CD spectroscopy and ESI-MS pH titrations show a distinct biphasic demetalation reaction, which we propose results from the presence of two metal–thiolate binding domains. We propose stoichiometries of Cd 3Cys 9 and Cd 4Cys 11 based on the presence of 20 cysteines split into two isolated regions of the sequence with 11 cysteines in the N-terminal and 9 cysteines in the C-terminal. The CD spectrum reported is distinctly different from any other metallothionein known suggesting quite different binding site structure for the peptide.

Reduced hippocampal MT2 melatonin receptor expression in Alzheimer's disease

The aim of the present study was to identify the distribution of the second melatonin receptor (MT2) in the human hippocampus of elderly controls and Alzheimer's disease (AD) patients. This is the first report of immunohistochemical MT2 localization in the human hippocampus both in control and AD cases. The specificity of the MT2 antibody was ascertained by fluorescence microscopy using the anti-MT2 antibody in HEK 293 cells expressing recombinant MT2, in immunoblot experiments on membranes from MT2 expressing cells, and, finally, by immunoprecipitation experiments of the native MT2. MT2 immunoreactivity was studied in the hippocampus of 16 elderly control and 16 AD cases. In controls, MT2 was localized in pyramidal neurons of the hippocampal subfields CA1-4 and in some granular neurons of the stratum granulosum. The overall intensity of the MT2 staining was distinctly decreased in AD cases. The results indicate that MT2 may be involved in mediating the effects of melatonin in the human hippocampus, and this mechanism may be heavily impaired in AD.