Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties

Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM₁ and AM₂ receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM₁ and AM₂ receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM₁ and AM₂ receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.     

Use of Cysteine Trapping to Map Spatial Approximations between Residues Contributing to the Helix N-capping Motif of Secretin and Distinct Residues within Each of the Extracellular Loops of Its Receptor

Amino-terminal regions of secretin-family peptides contain key determinants for biological activity and binding specificity, although the nature of interactions with receptors is unclear. A helix N-capping motif within this region has been postulated to directly contribute to agonist activity while also stabilizing formation of a helix extending toward the peptide carboxyl terminus and docking within the receptor amino terminus. We used cysteine trapping to systematically explore spatial approximations between cysteines replacing each residue in this motif of secretin (sec), Phe⁶, Thr⁷, and Leu¹⁰, and cysteines incorporated into the extracellular face of the receptor. Each peptide was a full agonist for cAMP, but had a lower binding affinity than natural hormone. These bound to COS cells expressing 61 receptor constructs incorporating cysteines in every position along each extracellular loop (ECL) and adjacent parts of transmembrane (TM) segments. Patterns of covalent labeling were distinct for each probe, with Cys⁶-sec labeling multiple residues in the carboxyl-terminal half of ECL2 and throughout ECL3, Cys⁷-sec predominantly labeling only single residues in the carboxyl-terminal end of ECL2 and the amino-terminal end of ECL3, and Cys¹⁰-sec not efficiently labeling any of these residues. These spatial constraints were used to refine our model of secretin bound to its receptor, now bringing ECL3 above the amino terminus of the ligand and revealing possible charge-charge interactions between this part of secretin and receptor residues in TM5, TM6, ECL2, and ECL3, which can orient and stabilize the peptide-receptor complex. This was validated by testing predicted approximations by mutagenesis and residue-residue complementation studies.     

Ric-8A,a GEF,and a Chaperone for G Protein α-Subunits: Evidence for the Two-Faced Interface

Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a prominent non-receptor GEF and a chaperone of G protein α-subunits (Gα). Recent studies shed light on the structure of Ric-8A, providing insights into the mechanisms underlying its interaction with Gα. Ric-8A is composed of a core armadillo-like domain and a flexible C-terminal tail. Interaction of a conserved concave surface of its core domain with the Gα C-terminus appears to mediate formation of the initial Ric-8A/GαGDP intermediate, followed by the formation of a stable nucleotide-free complex. The latter event involves a large-scale dislocation of the Gα α5-helix that produces an extensive primary interface and disrupts the nucleotide-binding site of Gα. The distal portion of the C-terminal tail of Ric-8A forms a smaller secondary interface, which ostensibly binds the switch II region of Gα, facilitating binding of GTP. The two-site Gα interface of Ric-8A is distinct from that of GPCRs, and might have evolved to support the chaperone function of Ric-8A.     

Identification of Distinct Conformations of the Angiotensin-II Type 1 Receptor Associated with the Gq/11 Protein Pathway and the β-Arrestin Pathway Using Molecular Dynamics Simulations

Biased signaling represents the ability of G protein-coupled receptors to engage distinct pathways with various efficacies depending on the ligand used or on mutations in the receptor. The angiotensin-II type 1 (AT₁) receptor, a prototypical class A G protein-coupled receptor, can activate various effectors upon stimulation with the endogenous ligand angiotensin-II (AngII), including the G_q/11 protein and β-arrestins. It is believed that the activation of those two pathways can be associated with distinct conformations of the AT₁ receptor. To verify this hypothesis, microseconds of molecular dynamics simulations were computed to explore the conformational landscape sampled by the WT-AT₁ receptor, the N111G-AT₁ receptor (constitutively active and biased for the G_q/11 pathway), and the D74N-AT₁ receptor (biased for the β-arrestin1 and -2 pathways) in their apo-forms and in complex with AngII. The molecular dynamics simulations of the AngII-WT-AT₁, N111G-AT₁, and AngII-N111G-AT₁ receptors revealed specific structural rearrangements compared with the initial and ground state of the receptor. Simulations of the D74N-AT₁ receptor revealed that the mutation stabilizes the receptor in the initial ground state. The presence of AngII further stabilized the ground state of the D74N-AT₁ receptor. The biased agonist [Sar¹,Ile⁸]AngII also showed a preference for the ground state of the WT-AT₁ receptor compared with AngII. These results suggest that activation of the G_q/11 pathway is associated with a specific conformational transition stabilized by the agonist, whereas the activation of the β-arrestin pathway is linked to the stabilization of the ground state of the receptor.     

Distinct second extracellular loop structures of the brain cannabinoid CB(1) receptor: implication in ligand binding and receptor function

The G-protein-coupled receptor (GPCR) second extracellular loop (E2) is known to play an important role in receptor structure and function. The brain cannabinoid (CB(1)) receptor is unique in that it lacks the interloop E2 disulfide linkage to the transmembrane (TM) helical bundle, a characteristic of many GPCRs. Recent mutation studies of the CB(1) receptor, however, suggest the presence of an alternative intraloop disulfide bond between two E2 Cys residues. Considering the oxidation state of these Cys residues, we determine the molecular structures of the 17-residue E2 in the dithiol form (E2(dithiol)) and in the disulfide form (E2(disulfide)) of the CB(1) receptor in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer, using a combination of simulated annealing and molecular dynamics simulation approaches. We characterize the CB(1) receptor models with these two E2 forms, CB(1)(E2(dithiol)) and CB(1)(E2(disulfide)), by analyzing interaction energy, contact number, core crevice, and cross correlation. The results show that the distinct E2 structures interact differently with the TM helical bundle and uniquely modify the TM helical topology, suggesting that E2 of the CB(1) receptor plays a critical role in stabilizing receptor structure, regulating ligand binding, and ultimately modulating receptor activation. Further studies on the role of E2 of the CB(1) receptor are warranted, particularly comparisons of the ligand-bound form with the present ligand-free form.     

Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function

The first and third extracellular loops (ECL) of G protein-coupled receptors (GPCRs) have been implicated in ligand binding and receptor function. This study describes the results of an alanine/leucine scan of ECLs 1 and 3 and loop-associated transmembrane (TM) domains of the secretin-like GPCR calcitonin receptor-like receptor which associates with receptor activity modifying protein 1 to form the CGRP receptor. Leu195Ala, Val198Ala and Ala199Leu at the top of TM2 all reduced αCGRP-mediated cAMP production and internalization; Leu195Ala and Ala199Leu also reduced αCGRP binding. These residues form a hydrophobic cluster within an area defined as the “minor groove” of rhodopsin-like GPCRs. Within ECL1, Ala203Leu and Ala206Leu influenced the ability of αCGRP to stimulate adenylate cyclase. In TM3, His219Ala, Leu220Ala and Leu222Ala have influences on αCGRP binding and cAMP production; they are likely to indirectly influence the binding site for αCGRP as well as having an involvement in signal transduction. On the exofacial surfaces of TMs 6 and 7, a number of residues were identified that reduced cell surface receptor expression, most noticeably Leu351Ala and Glu357Ala in TM6. The residues may contribute to the RAMP1 binding interface. Ile360Ala impaired αCGRP-mediated cAMP production. Ile360 is predicted to be located close to ECL2 and may facilitate receptor activation. Identification of several crucial functional loci gives further insight into the activation mechanism of this complex receptor system and may aid rational drug design.