Structural-functional analysis of the third transmembrane domain of the corticotropin-releasing factor type 1 receptor: role in activation and allosteric antagonism.

The corticotropin-releasing factor (CRF) type 1 receptor (CRF1R) for the 41-amino acid peptide CRF is a class B G protein-coupled receptor, which plays a key role in the response of our body to stressful stimuli and the maintenance of homeostasis by regulating neural and endocrine functions. CRF and related peptides, such as sauvagine, bind to the extracellular regions of CRF1R and activate the receptor. In contrast, small nonpeptide antagonists, which are effective against stress-related disorders, such as depression and anxiety, have been proposed to interact with the helical transmembrane domains (TMs) of CRF1R and allosterically antagonize peptide binding and receptor activation. Here, we aimed to elucidate the role of the third TM (TM3) in the molecular mechanisms underlying activation of CRF1R. TM3 was selected because its tilted orientation, relative to the membrane, allows its residues to establish key interactions with ligands, other TM helices, and the G protein. Using a combination of pharmacological, biochemical, and computational approaches, we found that Phe-203(3.40) and Gly-210(3.47) in TM3 play an important role in receptor activation. Our experimental findings also suggest that Phe-203(3.40) interacts with nonpeptide antagonists.

Exploring the binding site crevice of a family B G protein-coupled receptor, the type 1 corticotropin releasing factor receptor.

Family B of G protein-coupled receptors (GPCRs) is composed of receptors that bind peptides, such as secretin, glucagon, parathyroid hormone, and corticotropin releasing factor (CRF), which play critical physiological roles. These receptors, like all GPCRs, share a common structural motif of seven membrane-spanning segments, which have been proposed to bind small ligands, such as antalarmin, a nonpeptide antagonist of the type 1 receptor for CRF (CRF(1)). This leads to the hypothesis that as for family A GPCRs, the binding sites of small ligands for family B GPCRs are on the surface of a water-accessible crevice, the binding-site crevice, which is formed by the membrane-spanning segments and extends from the extracellular surface of the receptor into the plane of the membrane. To test this hypothesis we have begun to obtain structural information about family B GPCRs, using as a prototype the CRF(1), by determining the ability of sulfhydryl-specific methanethiosulfonate derivatives, such as the methanethiosulfonate-ethylammonium (MTSEA), to react with CRF(1) and thus irreversibly inhibit (125)I-Tyr(0)-sauvagine binding. We found that MTSEA inhibited (125)I-Tyr(0)-sauvagine binding to CRF(1) and that antalarmin protected against this irreversible inhibition. To identify the susceptible cysteine(s), we mutated, one at a time, four endogenous cysteines to serine. Mutation to serine of Cys211, Cys233, or Cys364 decreased the susceptibility of sauvagine binding to irreversible inhibition by MTSEA. Thus, Cys211, Cys233, and Cys364 at the cytoplasmic ends of the third, fourth, and seventh membrane-spanning segments, respectively, are exposed in the binding site crevice of CRF(1).

Synergistic contributions of the functional groups of epinephrine to its affinity and efficacy at the beta2 adrenergic receptor.

The structural basis of ligand affinity can be approached by studying the interactions between a drug and receptor residues; the basis for efficacy is more complex and must involve activation-associated conformational changes. We have used wild-type (WT), a constitutively active mutant (CAM), and a "constitutively inactive" mutant beta2 adrenergic receptor (beta(2)AR) to investigate changes in the binding site that accompany binding and activation. The active state (R(*)) probably involves repositioning of at least some of the agonist-contact residues, thereby optimizing their interactions with agonist and resulting in a higher affinity for agonist. A comparison of the binding affinities of a series of phenethylamine derivatives for WT revealed a remarkable synergism between the various functional groups present in epinephrine. Binding affinity was essentially unchanged with addition of beta-OH, N-CH(3), or catechol OHs to phenethylamine. In contrast, when each of these same groups was added to the appropriate compound, already containing the other two groups, to make epinephrine, the increase in affinity was quite large (60- to 120-fold). An initial interaction between two or more contacts may stabilize an intermediate conformation of beta(2)AR, R', either by altering amino acid side chain rotamer conformations or by a more global conformational change involving the repositioning of transmembrane segments. The pattern of these effects was different in the CAM in that fewer interactions were required to observe the synergistic effect, consistent with the hypothesis that the CAM mutation enriches the proportion of receptors in R(*) or in R' from which R(*) is more readily assumed.