Subtype-Specific Differences in Corticotropin-Releasing Factor Receptor Complexes Detected by Fluorescence Spectroscopy

Abstract

G protein-coupled receptors have been proposed to exist in signalosomes subject to agonist-driven shifts in the assembly disassembly equilibrium, affected by stabilizing membrane lipids and/or cortical actin restricting mobility. We investigated the highly homologous corticotropin-releasing factor receptors (CRFRs), CRFR1 and -2, which are different within their hydrophobic core. Agonist stimulation of CRFR1 and CRFR2 gave rise to similar concentration-response curves for cAMP accumulation, but CRFR2 underwent restricted collision coupling. Both CRFR1 and CRFR2 formed constitutive oligomers at the cell surface and recruited ␤-arrestin upon agonist activation (as assessed by fluorescence resonance energy transfer microscopy in living cells). However, CRFR2, but not CRFR1, failed to undergo agonist-induced internalization. Likewise, agonist binding accelerated the diffusion rate of CRFR2 only (detected by fluorescence recovery after photobleaching and fluorescence correlation spectroscopy) but reduced the mobile fraction, which is indicative of local confinement. Fluorescence intensity distribution analysis demonstrated that the size of CRFR complexes was not changed. Disruption of the actin cytoskeleton abolished the agonist-dependent increase in CRFR2 mobility, shifted the agonist concentration curve for CRFR2 to the left, and promoted agonist-induced internalization of CRFR2. Our observations are incompatible with an agonist-induced change in monomer-oligomer equilibrium, but they suggest an agonist-induced redistribution of CRFR2 into a membrane microdomain that affords rapid diffusion but restricted mobility and that is stabilized by the actin cytoskeleton. Our data show that membrane anisotropy can determine the shape and duration of receptor-generated signals in a subtypespecific manner.Signal transduction via heterotrimeric G proteins is accomplished by a cycle of activation and deactivation of the G␣-subunit, which is achieved by receptor-catalyzed exchange of prebound GDP for GTP and GTP hydrolysis by the intrinsic GTPase of G␣, respectively. Superimposed on this GTPase cycle, there is a cycle of subunit dissociation and reassociation, in which the inactive heterotrimer G␣.GDP.␤␥ affords receptor docking, GTP binding drives subunit dissociation into G␣.GTP.Mg 2ϩ and G␤␥, and the GTPase-mediated hydrolysis promotes mutual inactivation of two G␣.GDP and G␤␥ by reassociation of the inactive heterotrimer G␣.GDP.␤␥. This model was established some 20 years ago, mainly by the study of reconstituted purified components (Freissmuth et al., 1989). However, since then, methods have become available that allow the tracking of the activity of individual components at the single-cell level. In several instances, these have led to observations that are incompat-

The Specific Monomer/Dimer Equilibrium of the Corticotropin-releasing Factor Receptor Type 1 Is Established in the Endoplasmic Reticulum

“…3) Our result that the M/D of the CRF 1 R remains constant upon receptor activation is consistent with previous suggestions for this receptor using FRET experiments (29,30). This result may be specific for the CRF 1 R. However, the recently published smTIRFM studies for the N-formyl peptide receptor (25) and the ␤1-and ␤2-adrenergic receptors (26) also showed that the M/Ds of these receptors are unaffected by receptor activation.…”

“…Such a codiffusion would lead to a strong increase in diffusion time, which was not observed in our experiments. The detected diffusion times were consistent with the literature (30), and the cross-correlation amplitudes derived from the FCCS experiments thus only reflect receptor interactions. In the case of the FCCS measurements at the ER membrane, however, we observed in some experiments an increase in diffusion time to a certain extent.…”

“…As class B receptors, we took the CRF 1 R and the CRF 2(a) R. All these receptors were described to form dimers (14,17,29,30,(43)(44)(45) with the exception of the CRF 2(a) R, which is expressed exclusively as a monomer (14). For FCCS, the full-length receptors were C-terminally fused to GFP or mCherry.…”

“…This single molecule-based method was applied successfully to monitor GPCR dimerization (14,27,28). As a model, we took a class B receptor, namely the CRF 1 R, which forms dimers (14,29,30) and is expressed mainly in the anterior pituitary where it plays a central role in the regulation of the hypothalamic-pituitary-adrenal stress axis in mammals (31). As a control, we took the CRF 2(a) R, which forms exclusively monomers due to the presence of a unique domain at the N tail of the receptor, its pseudo signal peptide (14).…”

Fluorescence/Bioluminescence Resonance Energy Transfer Techniques to Study G-Protein-Coupled Receptor Activation and Signaling

“…Likewise, Pontier et al (2008) proposed that ␤ 2 -adrenergic receptors did not partition within the liquid-ordered lipid phase, whereas G-proteins and adenylyl cyclases were sequestered in these domains, and that their mobilization led to increased receptor-mediated signaling. Milan-Lobo et al (2009) suggested, on the basis of FRET and FRAP studies, that the restricted mobility of corticotropin-releasing factor receptors (CRFR2) may be increased by agonist activation of the receptors, suggesting a transfer into a different membrane compartment or loss of contact with the cytoskeleton.…”