Receptor activity-modifying protein-dependent effects of mutations in the calcitonin receptor-like receptor:implications for adrenomedullin and calcitonin gene-related peptide pharmacology

Background and Purpose Receptor activity-modifying proteins (RAMPs) define the pharmacology of the calcitonin receptor-like receptor (CLR). The interactions of the different RAMPs with this class B GPCR yield high-affinity calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors. However, the mechanism for this is unclear. Experimental Approach Guided by receptor models, we mutated residues in the N-terminal helix of CLR, RAMP2 and RAMP3 hypothesized to be involved in peptide interactions. These were assayed for cAMP production with AM, AM2 and CGRP together with their cell surface expression. Binding studies were also conducted for selected mutants. Key Results An important domain for peptide interactions on CLR from I32 to I52 was defined. Although I41 was universally important for binding and receptor function, the role of other residues depended on both ligand and RAMP. Peptide binding to CLR/RAMP3 involved a more restricted range of residues than that to CLR/RAMP1 or CLR/RAMP2. E101 of RAMP2 had a major role in AM interactions, and F111/W84 of RAMP2/3 was important with each peptide. Conclusions and Implications RAMP-dependent effects of CLR mutations suggest that the different RAMPs control accessibility of peptides to binding residues situated on the CLR N-terminus. RAMP3 appears to alter the role of specific residues at the CLR-RAMP interface compared with RAMP1 and RAMP2

Functional solubilization of the β 2-adrenoceptor using diisobutylene maleic acid

The β2-adrenoceptor (β2AR) is a well-established target in asthma and a prototypical G protein-coupled receptor for biophysical studies. Solubilization of membrane proteins has classically involved the use of detergents. However, the detergent environment differs from the native membrane environment and often destabilizes membrane proteins. Use of amphiphilic copolymers is a promising strategy to solubilize membrane proteins within their native lipid environment in the complete absence of detergents. Here we show the isolation of the β 2AR in the polymer diisobutylene maleic acid (DIBMA). We demonstrate that β 2AR remains functional in the DIBMA lipid particle and shows improved thermal stability compared with the n-dodecyl-β-D-maltopyranoside detergent-solubilized β 2AR. This unique method of extracting β 2AR offers significant advantages over previous methods routinely employed such as the introduction of thermostabilizing mutations and the use of detergents, particularly for functional biophysical studies

Constraining the evolution of cataclysmic variables via the masses and accretion rates of their underlying white dwarfs

We report on the masses (MWD), effective temperatures (⁠Teff⁠) and secular mean accretion rates (⁠⟨M˙⟩⁠) of 43 cataclysmic variable (CV) white dwarfs, 42 of which were obtained from the combined analysis of their Hubble Space Telescope ultraviolet data with the parallaxes provided by the Early Third Data Release of the Gaia space mission, and one from the white dwarf gravitational redshift. Our results double the number of CV white dwarfs with an accurate mass measurement, bringing the total census to 89 systems. From the study of the mass distribution, we derive ⟨MWD⟩=0.81+0.16/−0.20M⊙⁠, in perfect agreement with previous results, and find no evidence of any evolution of the mass with orbital period. Moreover, we identify five systems with MWD < 0.5M⊙, which are most likely representative of helium-core white dwarfs, showing that these CVs are present in the overall population. We reveal the presence of an anti-correlation between the average accretion rates and the white dwarf masses for the systems below the 2 − 3 h period gap. Since ⟨M˙⟩ reflects the rate of system angular momentum loss, this correlation suggests the presence of an additional mechanism of angular momentum loss that is more efficient at low white dwarf masses. This is the fundamental concept of the recently proposed empirical prescription of consequential angular momentum loss (eCAML) and our results provide observational support for it, although we also highlight how its current recipe needs to be refined to better reproduce the observed scatter in Teff and ⟨M˙⟩⁠, and the presence of helium-core white dwarfs