Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.6 (thromboplastin)
 13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Each member of the muscarinic receptor family (M1-M5) can interact only with a limited subset of the many structurally closely related heterotrimeric G proteins expressed within a cell. To understand how this selectivity is achieved at a molecular level, we have used the G(i/0)-coupled M2 and the Gq/11-coupled M3 muscarinic receptors as model systems. We developed a genetic strategy involving the coexpression of wild type or mutant muscarinic receptors with hybrid or mutant G protein alpha subunits to identify specific, functionally relevant receptor/G protein contact sites. This approach led to the identification of N- and C-terminal amino acids on alpha(q) and alpha(i) that are critical for maintaining proper receptor/G protein coupling. Moreover, several receptor sites were identified that are likely to be contacted by these functionally critical G alpha residues. To gain deeper insight into muscarinic receptor structure, we recently developed a cysteine disulfide cross-linking strategy, using the M3 muscarinic receptor as a model system. Among other structural modifications, this approach involves the removal of most native cysteine residues by site-directed mutagenesis, the insertion of three factor Xa cleavage sites into the third intracellular loop, and systematic 'reintroduction' of pairs of cysteine residues. Following treatment of receptor-containing membrane preparations with factor Xa and oxidizing agents, disulfide cross-linked products can be identified by immunoprecipitation and immunoblotting studies. This approach should greatly advance our knowledge of the molecular architecture of muscarinic and other G protein-coupled receptors.
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PMID:Structure-function analysis of muscarinic receptors and their associated G proteins. 1006 96

The structural changes involved in ligand-dependent activation of G protein-coupled receptors are not well understood at present. To address this issue, we developed an in situ disulfide cross-linking strategy using the rat M(3) muscarinic receptor, a prototypical G(q)-coupled receptor, as a model system. It is known that a tyrosine residue (Tyr(254)) located at the C terminus of transmembrane domain (TM) V and several primarily hydrophobic amino acids present within the cytoplasmic portion of TM VI play key roles in determining the G protein coupling selectivity of the M(3) receptor subtype. To examine whether M3 receptor activation involves changes in the relative orientations of these functionally critical residues, pairs of cysteine residues were substituted into a modified version of the M(3) receptor that contained a factor Xa cleavage site within the third intracellular loop and lacked most endogenous cysteine residues. All analyzed mutant receptors contained a Y254C point mutation and a second cysteine substitution within the segment Lys(484)-Ser(493) at the intracellular end of TM VI. Following their transient expression in COS-7 cells, mutant receptors present in their native membrane environment (in situ) were subjected to mild oxidizing conditions, either in the absence or in the presence of the muscarinic agonist, carbachol. The successful formation of disulfide cross-links was monitored by studying changes in the electrophoretic mobility of oxidized, factor Xa-treated receptors on SDS gels. The observed cross-linking patterns indicated that M(3) receptor activation leads to structural changes that allow the cytoplasmic ends of TM V and TM VI to move closer to each other and that also appear to involve a major change in secondary structure at the cytoplasmic end of TM VI. This is the first study employing an in situ disulfide cross-linking strategy to examine agonist-dependent dynamic structural changes in a G protein-coupled receptor.
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PMID:Conformational changes that occur during M3 muscarinic acetylcholine receptor activation probed by the use of an in situ disulfide cross-linking strategy. 1169 1

In this study, we employed an in situ disulfide cross-linking strategy to gain insight into the structure of the inactive and active state of the M(3) muscarinic acetylcholine receptor. Specifically, this study was designed to identify residues in TM I that are located in close to Cys532 (position 7.42), an endogenous cysteine residue present in the central portion of TM VII. Cysteine residues were substituted, one at a time, into 10 consecutive positions of TM I (Ala71-Val80) of a modified version of the M(3) muscarinic receptor that lacked most endogenous cysteine residues and contained a factor Xa cleavage site within the third intracellular loop. Following their expression in COS-7 cells, the 10 resulting cysteine mutant receptors were oxidized in their native membrane environment, either in the absence or in the presence of muscarinic ligands. Disulfide cross-link formation was monitored by examining changes in the electrophoretic mobility of oxidized and factor Xa-digested receptors on SDS gels. When molecular iodine was used as the oxidizing agent, the L77C receptor (position 1.42) was the only mutant receptor that displayed significant disulfide cross-linking, either in the absence or in the presence of muscarinic agonists or antagonists. On the other hand, when the Cu(II)-(1,10-phenanthroline)(3) complex served as the redox catalyst, muscarinic ligands inhibited disulfide cross-linking of the L77C receptor, probably because of impaired access of this relatively bulky oxidizing agent to the ligand binding crevice. The iodine cross-linking data suggest that M(3) muscarinic receptor activation is not associated with significant changes in the relative orientations of the outer and/or central segments of TM I and VII. In bovine rhodopsin, the residues present at the positions corresponding to Cys532 and Leu77 in the rat M(3) muscarinic receptor are not located directly adjacent to each other, raising the possibility that the relative orientations of TM I and VII are not identical among different class I GPCRs. Alternatively, dynamic protein backbone fluctuation may occur, enabling Cys532 to move within cross-linking distance of Leu77 (Cys77).
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PMID:Use of an in situ disulfide cross-linking strategy to map proximities between amino acid residues in transmembrane domains I and VII of the M3 muscarinic acetylcholine receptor. 1205 96

We studied the role of carboxyl tail cysteine residues and their palmitoylation in constitutive signaling by the thyrotropin-releasing hormone (TRH) receptor type 1 (TRH-R1) in transfected mammalian cells and in Xenopus laevis oocytes. To study palmitoylation, we inserted a factor Xa cleavage site within the third extracellular loop of TRH-R1, added a carboxyl-terminal C9 immunotag and expressed the mutant receptor in Chinese hamster ovary cells. We identified TRH-R1-specific palmitoylation in the transmembrane helix-7/carboxyl-tail receptor fragment mainly at Cys-335 and Cys-337. In contrast to a mutant truncated at Cys-335 that was reported previously to be constitutively active, a receptor truncated at Lys-338 (K338Stop), which preserves Cys-335 and Cys-337, and C337Stop and N336Stop, which preserve Cys-335, did not exhibit increased constitutive signaling. TRH-R1 mutants substituted singly by Gly or Ser at Cys-335 or Cys-337 did not exhibit constitutive signaling. By contrast, substitution of both cysteines (C335G/C337G or C335S/C337S) yielded TRH-R1 mutants that exhibited marked constitutive signaling in mammalian cells. In the oocyte, constitutive signaling by C335G/C337G resulted in homologous (of C335G/C337G) and heterologous (of M1 muscarinic receptor) desensitization. Because both Cys-335 and Cys-337 have to be substituted or deleted for constitutive signaling, we propose that a single palmitoylation site in the proximal carboxyl tail is sufficient to constrain TRH-R1 in an inactive conformation.
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PMID:Carboxyl tail cysteine mutants of the thyrotropin-releasing hormone receptor type 1 exhibit constitutive signaling: role of palmitoylation. 1583 33