Serotonin 5-HT 4(a) receptor, a G-protein-coupled receptor (GPCR), was produced as a functional isolated protein using Escherichia coli as an expression system. The isolated receptor was characterized at the molecular level by circular dichroism (CD) and steady-state fluorescence. A specific change in the near-UV CD band associated with the GPCR disulfide bond connecting the third transmembrane domain to the second extracellular loop (e2) was observed upon agonist binding to the purified receptor. This is a direct experimental evidence for a change in the conformation of the e2 loop upon receptor activation. Different variations were obtained depending whether the ligand was an agonist (partial or full) or an inverse agonist. In contrast, antagonist binding did not induce any variation. These observations provide a first direct evidence for the fact that free (or antagonist-occupied), active (partial-or full agonist-occupied) and silent (inverse agonist-occupied) states of the receptor involve different arrangements of the e2 loop. Finally, ligand-induced changes in the fluorescence emission profile of the purified receptor confirmed that the partial agonist stabilized a single, welldefined, conformational state and not a mixture of different states. This result is of particular interest in a pharmacological perspective since it directly demonstrates that the efficacy of a drug is likely due to the stabilization of a ligand-specific state rather than selection of a mixture of different conformational states of the receptor.G-protein-coupled receptors are versatile biological sensors. They are responsible for the majority of cellular responses to hormones and neurotransmitters, as well as sight, smell, and taste senses (1, 2). Signal transduction is specifically associated with GPCR 1 activation. Although significant progress has been made within the last few years in dissecting GPCR-mediated signal transduction pathways, understanding of the mechanisms underlying receptor activation is still hampered by the lack of information at the molecular level (3,4). This is largely due to the fact that very few expression systems have proven satisfactory for producing these receptors in a functional state and with sufficient yields for biophysical studies to be carried out (5-7). Most of the systems that have been developed to elucidate the mechanism of GPCR activation therefore essentially rely on the use of purified rhodopsin and ␤ 2 -adrenergic receptor (3,4,8). Interestingly, most of the results obtained so far report on the conformational events occurring at the level of the cytoplasmic side of the receptors. In contrast, few reports give indications on the possible conformational rearrangements certainly occurring in the extracellular part of the receptor, in particular in the extracellular loops.Several models have been developed to conceptualize the mechanisms of activation (9, 10). The two-state model and the extended ternary model assumes that the receptor exists in an equilibrium between a resting state (R) and...

G-protein-coupled receptors (GPCRs) are key players in cell communication. Although long considered as monomeric, it now appears that these heptahelical proteins can form homo-or heterodimers. Here, we analyzed the conformational changes in each subunit of a receptor dimer resulting from agonist binding to either one or both subunits by measuring the fluorescent properties of a leukotriene B 4 receptor dimer with a single 5-hydroxytryptophan-labeled protomer. We show that a receptor dimer with only a single agonist-occupied subunit can trigger G-protein activation. We also show that the two subunits of the receptor dimer in the G-protein-coupled state differ in their conformation, even when both are liganded by the agonist. No such asymmetric conformational changes are observed in the absence of G-protein, indicating that the interaction of the G-protein with the receptor dimer brings specific constraints that prevent a symmetric functioning of this dimer. These data open new options for the differential signaling properties of GPCR dimers.

We have used an isolated receptor, the leukotriene B 4 receptor BLT1, to analyze the mechanism of receptor activation in a G-protein-coupled receptor dimer. The isolated receptor is essentially a dimer whether the agonist is present or not, provided the detergent used stabilizes the inactive dimeric assembly. We have produced a receptor mutant where Cys 97 in the third transmembrane domain has been replaced by a serine. This mutation leads to an ϳ100-fold decrease in the affinity for the agonist. 5-Hydroxytryptophan has then been introduced at position 234 in the C97A mutant sixth transmembrane domain. Agonist binding to the labeled receptor is associated with variations in the fluorescence properties of 5-hydroxytryptophan due to specific agonist-induced conformational changes. The C97A mutant labeled with 5-hydroxytryptophan has then been associated with a wild-type receptor in a dimeric complex that has been subsequently purified. The purified complex activates its G-protein partner in a similar manner as the wildtype homodimer. Due to the difference in the affinity for the agonist between the wild-type and mutant protomers in this dimer, we have been able to reach a state where one of the protomers, the mutant, is in its unliganded state, whereas the other, the wild type, is loaded with the agonist. We show that agonist binding to the wild-type receptor induces specific changes in the conformation of the unliganded protomer, as evidenced by the variations in the emission of the 5-hydroxytryptophan residue in the mutant receptor. These data provide a direct demonstration for agonist-induced cooperative conformational changes in a GPCR dimer.

Rat liver chromatin core particles digested with clostripain yield a structurally well‐defined nucleoprotein particle with an octameric core made up of fragmented histone species (designated H'2A, H'2B, H'3 and H'4, respectively) after selective loss of a sequence segment located in the N‐terminal region of each core histone. Sequential Edman degradation and carboxypeptidase digestion unambiguously establish that histones H2A, H2B, H3 and H4 are selectively cleaved at the carboxyl side of Arg 11, Lys 20, Arg 26 and Arg 19 respectively and that the C‐terminal sequences remain unaffected. Despite the loss of the highly basic N‐terminal regions, including approximately 17% of the total amino acids, the characteristic structural organization of the nucleosome core particle appears to be fully retained in the proteolyzed core particle, as judged by physicochemical and biochemical evidence. Binding of spermidine to native and proteolyzed core particles shows that DNA accessibility differs markedly in both structures. As expected the proteolyzed particle, which has lost all the in vivo acetylation sites, is not enzymatically acetylated, in contrast to the native particle. However, proteolyzed histones act as substrates of the acetyltransferase in the absence of DNA, as a consequence of the occurrence of potential acetylation sites in the core histones thus rendered accessible. The possible role of the histone N‐terminal regions on chromatin structure and function is discussed in the light of the present observations with the new core particle obtained by clostripain proteolysis.

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To assess the role of each of the four cysteine residues in the hormone binding domain (HBD) of the human mineralocorticoid receptor (hMR), we have separately substituted C808, C849, C910, and C942 into serine. These mutations were created in the G595-K984 hMR receptor fragment which encompasses the DNA binding domain, the hinge region, and the hormone binding domain. Each mutant was further analyzed by steroid binding assays and transactivation assays using wild-type and mutant proteins expressed in vitro in the reticulocyte lysate. While the C910S mutant exhibited similar wild-type G595-K984 receptor binding properties for aldosterone, the C808S mutant affinity was 3.5-fold higher. In contrast, the C849S mutant showed a drastic drop in affinity for aldosterone and the mutant C942S was unable to bind the steroid. Affinities for the antagonist progesterone were also determined. C808S, C849S, and C910S were found to bind progesterone better than aldosterone (about a 2-fold increase in their affinities). Mutant C942S failed to bind any steroid tested (aldosterone, progesterone, cortisol, and the synthetic antagonist RU26752). No change in the specificity toward various agonists and antagonists could be observed with the mutants when compared to the wild-type G595-K984. When transactivation assays were performed, the properties of mutants C808S and C910S were similar to those of the wild-type G595-K984, while mutant C849S showed reduced sensitivity and C942S was devoid of any transcriptional activity. Our data indicate that C849 and C942 are critical for the ligand binding process of hMR. Moreover, C942 might be involved in a direct contact with the 20-keto group of the steroid.

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