No abstract

Site‐directed mutagenesis and molecular dynamics simulations of the alpha 1B‐adrenergic receptor (AR) were combined to explore the potential molecular changes correlated with the transition from R (inactive state) to R (active state). Using molecular dynamics analysis we compared the structural/dynamic features of constitutively active mutants with those of the wild type and of an inactive alpha 1B‐AR to build a theoretical model which defines the essential features of R and R. The results of site‐directed mutagenesis were in striking agreement with the predictions of the model supporting the following hypothesis. (i) The equilibrium between R and R depends on the equilibrium between the deprotonated and protonated forms, respectively, of D142 of the DRY motif. In fact, replacement of D142 with alanine confers high constitutive activity to the alpha 1B‐AR. (ii) The shift of R143 of the DRY sequence out of a conserved ‘polar pocket’ formed by N63, D91, N344 and Y348 is a feature common to all the active structures, suggesting that the role of R143 is fundamental for mediating receptor activation. Disruption of these intramolecular interactions by replacing N63 with alanine constitutively activates the alpha 1B‐AR. Our findings might provide interesting generalities about the activation process of G protein‐coupled receptors.

According to classical models of drugreceptor interactions, competitive antagonists share with agonists the ability to bind to a common site on the receptor molecule. However, they are different from agonists, as they cannot trigger the "stimulus" that leads to biological responses-i.e., they lack intrinsic activity. For those receptors whose signals are transduced to effector systems by GTPbinding regulatory proteins (G proteins), a mechanistic equivalent of such a stimulus is an increased ability of agonist-bound receptor to accelerate nucleotide exchange and thus GTPase activity on the G-protein molecule. Here we show that for a member of this family of receptors (6 opioid receptors in membranes of NG108-15 neuroblastoma-glioma cells), two types of competitive antagonists can be distinguished. One type has no intrinsic activity, since it neither stimulates nor inhibits the GTPase activity of G proteins and its apparent ainmity for the receptor is not altered by pertussis toxin-mediated uncoupling of receptor and G protein. The second type, however, can inhibit GTPase and thus exhibits negative intrinsic activity; its affinity for receptors is increased following uncoupling from G proteins. The existence of antagonists with negative intrinsic activity may be a general feature of several classes of neurotransmitters or hormone receptors and calls for a reevaluation of biological effects produced by competitive antagonists.Although ,u and 8 opioid receptors can be clearly distinguished on a pharmacological basis (1), recent evidence (2, 3) indicates that these two types of receptors share the ability to interact with GTP-binding regulatory proteins (G proteins). In this respect, they belong to a large family of hormone and neurotransmitter receptors whose signals are transmitted to enzymes and ion channels across plasma membranes by intervening G proteins (reviews in refs. 4-6). Activation of one or more G proteins, which results in increase of GTPase activity, is the first detectable biochemical event that follows recognition of this group of receptors by agonists, regardless of the sort of signal that is actually propagated to effector molecules (5, 6). Receptor-mediated activation of G proteins involves the establishment of a ternary complex between ligand-occupied receptor and G protein, as suggested long before the isolation of G proteins. The findings (i) that guanine nucleotides exert negative heterotropic effects on the affinity of the receptor (7) only when the receptor is occupied by an agonist (8, 9), (ii) that a receptor prelabeled by agonists can be solubilized in a higher molecular weight form than when prelabeled by antagonists (10), and (iii) that agonists but not antagonists display complex binding isotherms in the absence of guanine nucleotides (11) liposomes (12, 13). Accordingly, the intrinsic activities of receptor ligands represent their ability to stabilize the ternary complex and range from null values for antagonists, which passively occupy the binding site, to various degrees of...

G-protein coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signaling effectors such as G proteins and β-arrestins. It is widely appreciated that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (i.e., ‘efficacy’)1. Furthermore, mounting biophysical evidence, primarily using the β-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states2–5. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct β2AR conformations using single domain camelid antibodies (nanobodies): a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60)6,7. We show that Nb60 stabilizes a previously unappreciated low affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoproterenol has a 15,000-fold higher affinity for the β2AR in the presence of Nb80 compared to Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states.

In this study, a quantitative approach was used to investigate the role of D142, which belongs to the highly conserved E͞DRY sequence, in the activation process of the ␣ 1B -adrenergic receptor (␣ 1B -AR). Experimental and computer-simulated mutagenesis were performed by substituting all possible natural amino acids at the D142 site. The resulting congeneric set of proteins together with the finding that all the receptor mutants show various levels of constitutive (agonist-independent) activity enabled us to quantitatively analyze the relationships between structural͞dynamic features and the extent of constitutive activity. Our results suggest that the hydrophobic͞hydrophilic character of D142, which could be regulated by protonation͞deprotonation of this residue, is an important modulator of the transition between the inactive (R) and active (R*) state of the ␣ 1B -AR. Our study represents an example of quantitative structureactivity relationship analysis of the activation process of a G protein-coupled receptor.The ␣ 1B -adrenergic receptor (AR) belongs to the superfamily of G protein-coupled receptors (GPCRs). The seven transmembrane domains (TMDs) common to all GPCRs contribute to the formation of the ligand binding pocket, whereas aa sequences of the intracellular loops (i) appear to mediate receptor-G protein coupling (1, 2). However, how binding of the extracellular signals is converted into receptor activation remains largely unknown.Recently, we have investigated the activation process of the ␣ 1B -AR linked to phospholipase C-mediated activation of polyphosphoinositide hydrolysis. By a combination of site-directed mutagenesis of the ␣ 1B -AR with computational simulations of receptor dynamics we explored the potential molecular mechanisms underlying the process of receptor activation, by focusing on a number of constitutively active receptor mutants (3). We identified a series of molecular changes that appear to be correlated with the transition from the inactive (R) to the active (R*) state, independently of the presence of the agonist. We proposed that the equilibrium between R and R* of the ␣ 1B -AR depends, at least in part, on the prototropic equilibrium between the deprotonated (anionic) and protonated (neutral) forms of D142, the negatively charged residue present in the E͞DRY motif, which is highly conserved among different GPCRs (Fig. 1A). As a result, we found that replacement of D142 with the nonpolar aa alanine conferred high constitutive (agonist-independent) activity to the ␣ 1B -AR. According to our analysis, a series of intramolecular interactions that might be of fundamental importance in the process of receptor activation depends on the protonation state of D142. In particular, our model pointed to a conserved ''polar pocket'' formed near the cytosol via a network of Hbonding interactions among N63, D91, N344, and Y348 (Fig. 1 A). This set of interactions constrains the receptor in its inactive state by exerting control on the degree of cytosolic exposure of the arginine resid...