| 1. |
- Fu, Michael, 1963, et al.
(författare)
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Agonist-like activity of anti-peptide antibodies directed against an autoimmune epitope on the heart muscarinic acetylcholine receptor.
- 1994
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Ingår i: Receptors & channels. - 1060-6823. ; 2:2, s. 121-30
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Tidskriftsartikel (refereegranskat)abstract
- A synthetic peptide corresponding to amino acids 169-193 of the second extracellular loop of the M2 human muscarinic receptor was used to raise antibodies in rabbits. Affinity purified antibodies specifically recognized a major band with a molecular weight of about 80 kDa on the electrotransferred membrane proteins of both rat ventricles and chinese hamster ovary cells expressing recombinant muscarinic receptors. Incubation of these antibodies with rat myocardial membranes resulted not only in a decrease in the maximal binding capacity, but also in a decrease in receptor antagonist affinity. These antibodies could also mimic the effects of agonist stimulation as demonstrated by inhibition of isoproterenol-stimulated cAMP accumulation and by a negative chronotropic effect on cultured cardiomyocytes. These results suggest that the second extracellular loop of the M2 muscarinic receptor is an immunologically and functionally important domain with properties comparable to those found for autoantibodies against the same domain in idiopathic dilated cardiomyopathy. It strengthens the hypothesis that the second extracellular loop of the members of the superfamily of G-protein coupled membrane receptors could be the main immunogenic region responsible for a pathogenic autoimmune response.
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| 2. |
- Fu, Michael, 1963, et al.
(författare)
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Immunohistochemical localization of angiotensin II receptors (AT1) in the heart with anti-peptide antibodies showing a positive chronotropic effect.
- 1998
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Ingår i: Receptors & channels. - 1060-6823. ; 6:2, s. 99-111
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies were produced against a synthetic peptide corresponding to amino acids (165-191) of the second extracellular loop of the human angiotensin II receptor subtype 1 (AT1) in rabbits. The purified antibodies had an apparent affinity of about 1 nM and were monospecific for the AT1-receptor peptide. Chemical modification of the carboxyl groups (glu at positions 173 and 185) and the sulfhydryl group (cys at position 180) of the AT1-receptor peptide did not alter the relative affinity of the coated AT1-receptor peptide to antibodies. The antibodies specifically stained CHO cells expressing the rat AT1a receptor. Immunoblots on rat kidney revealed that the antibody recognized a protein band of 59 +/- 3 kDa in a dose-dependent manner and this band was no longer detected after preincubating the antibodies with AT1-receptor peptide. Using electron microscopic and immunofluorescence immunocytochemistry techniques, angiotensin II receptors were detected in (1) the sarcolemma, T-tubules and nuclei of rat cardiomyocytes, (2) the transluminal side of endothelial cells and (3) fibroblast cells. These localizations are specific, as the immunostaining did not appear when preimmune rabbit serum was used and was blocked after preincubating antibodies with antigenic peptide. Functionally, these antibodies did not affect the ligand binding properties of the receptors but displayed agonist-like activity as shown by dose-dependent increases in beating frequency in cultured neonatal cardiomyocytes. These results suggest that the antibodies against the second extracellular loop of human AT1 receptors were able to specifically recognize AT1 receptors. In addition, they extend the observation that the second extracellular loop of the G-protein coupled membrane receptors is a specific target for antibodies with agonist-like activity.
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| 3. |
- Kukkonen, Jyrki
(författare)
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Regulation of receptor-coupling to (multiple) G-proteins. A challenge for basic research and drug discovery
- 2004
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Ingår i: Receptors Channels. - 1060-6823. ; 10:5-6, s. 167-83
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Tidskriftsartikel (refereegranskat)abstract
- G protein-coupled receptors induce intracellular signals via interaction of with cytosolic/peripheral membrane proteins, mainly G proteins. There has been much debate about the mode of interaction between the receptors, G proteins and effectors, their mobility and the ways of determining the specificity of interaction. Additional complexity has been added to system upon the discovery of i) coupling of single receptors to several G proteins and ii) active direction of this by different ligands (stimulus trafficking). These data suggest that the most primary unit in the signal transduction is the receptor complexed with a specific G protein, making the investigation of the mechanism of receptor–G protein selection and interaction even more important. In this review, I will summarize the general knowledge of receptor interaction with G proteins and effectors and the ways of investigating this.
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| 4. |
- Monstein, H.-J., et al.
(författare)
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Cloning and characterization of 5'-end alternatively spliced human cholecystokinin-B receptor mRNAs
- 1998
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Ingår i: Receptors and Channels. - 1060-6823 .- 1607-856X. ; 6:3, s. 165-177
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Tidskriftsartikel (refereegranskat)abstract
- We report here the cloning and characterization of a 5'-end alternatively spliced human cholecystokinin-B (CCK-B) receptor mRNA. The 5'-end of this CCK-B receptor transcript (termed CCK-BRtx) consisted of exon Ia, present in the ordinary full-length CCK-B receptor mRNA (CCK-BRwt), and exon Ib, present in a previously described 5'-end alternatively spliced CCK-B receptor mRNA (CCK-BRt). A short open reading frame preceded the AUG translation initiation codon of the CCK-BRtx. Transfection of COS-7 cells with the CCK-BRtx or CCK-BRt cDNAs did not lead to the appearance of peptidergic and non-peptidergic binding sites. Cell free in vitro translation yielded proteins of approximately 44 kDa (CCK-B receptor) and 40 kDa (CCK-BRt receptor) whereas no 40 kDa product was detected from the cloned CCK-BRtx cDNA. Instead, a protein product of approximately 9 kDa was visualized, the size corresponding to the predicted protein encoded by the short open reading frame. The alternatively spliced CCK-B receptor transcripts were concomitantly expressed with the ordinary full-length CCK-B receptor mRNA in the brain, pancreas, and stomach. The possibility that such transcripts are translated in vivo into truncated CCK-B receptors is discussed.
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| 5. |
- Tierney, M L, et al.
(författare)
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Two threonine residues in the M2 segment of the alpha 1 beta 1 GABAA receptor are critical for ion channel function.
- 1998
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Ingår i: Receptors and Channels. - 1060-6823 .- 1607-856X. ; 5:2, s. 113-24
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Tidskriftsartikel (refereegranskat)abstract
- The role of three threonine residues in the M2 hydrophobic region of the GABAA receptor has been investigated by replacing these polar residues with alanine at the 6', 10' and 13' positions of M2 in the GABAA alpha 1, and beta 1 subunits and co-expressing the mutated subunits in the baculovirus Sf9 insect cell system. GABA did not elicit a current in cells expressing either the 6' or 13' threonine to the alanine mutants. The mutant subunits formed intact heteromeric GABAA receptors as judged by the binding of [3H] muscimol or the relative level of alpha 1 protein present in the plasma membrane. In contrast, a chloride current was generated by GABA in cells expressing the 10' mutant receptor. However, the current decayed more rapidly to baseline in the continued presence of GABA in the 10' mutant receptor than in the wild type receptor. The results are discussed in terms of the possible roles of the threonine residues in the ion conduction pathway.
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