Constitutive formation of an RXFP1-signalosome: a novel paradigm in GPCR function and regulation

Abstract

The classical second messenger cAMP is important in diverse physiological processes, where its spatial and temporal compartmentalization allows precise control over multiple cellular events. Within this context, G-protein-coupled receptors (GPCRs) govern specialized pools of cAMP, which are functionally specific for the unique cellular effects attributed to a particular system. The relaxin receptor, RXFP1, is a GPCR that exerts pleiotropic physiological effects including a potent anti-fibrotic response, increased cancer metastases, and has efficacy as a vasodilator in heart failure. On a cellular level, relaxin stimulation of RXFP1 results in the activation of multiple G-protein pathways affecting cAMP accumulation. Specificity and diversity in the cAMP signal generated by RXFP1 is controlled by differential G-protein coupling dependent upon the background of cellular expression, and cAMP compartmentalization. Further complexity in cAMP signalling results from the constitutive assembly of an RXFP1-signalosome, which specifically responds to low concentrations of relaxin, and activates a distinct cAMP pathway. The RXFP1-signalosome is a higher-order protein complex that facilitates receptor sensitivity to attomolar concentration of peptide, exhibits constitutive activity and dual coupling to G-proteins and β-arrestins and reveals a concentration-biased agonism mediated by relaxin. The specific and directed formation of GPCR-centered signalosomes allows an even greater spatial and temporal control of cAMP, thus rationalizing the considerable physiological scope of this ubiquitous second messenger.

Figure 1

Concentration-biased signalling at the relaxin receptor, RXFP1, compared with prototypical β₂-adrenoceptor signalling. The relaxin receptor RXFP1 demonstrates differential activation of intracellular signalling pathways, leading to increased cAMP accumulation in response to increasing concentrations of ligand; this is in contrast to the prototypical activation, desensitization and internalization paradigm demonstrated by the β₂-adrenoceptor. A. Under basal conditions, expression of RXFP1 induces the formation of an active signalosome. AKAP79 interacts with helix 8 of the RXFP1 C-terminal tail and thereby scaffolds AC2 to the vicinity of the receptor; this allows efficient activation of AC2 by both Gα_s and Gβγ-subunits. The cAMP generated by the stimulation of AC2 is tightly controlled by the activity of PDE4D3. This phosphodiesterase is activated by PKA (in a negative feedback loop) and additionally interacts with β-arrestin 2, which binds to Ser⁷⁰⁴ of the RXFP1 C-terminus, thereby anchoring the regulatory proteins to the signalosome. B. Sub-picomolar concentrations of relaxin (down to attomolar levels) further activate the pre-assembled, receptor-driven signalosome. The cAMP generated following signalosome activation is maintained within a tightly defined range by the activity of PDE4D3, and this is probably complemented by direct AKAP79-mediated inhibition of AC2 activity. C. Increasing concentrations of relaxin appear to induce dissociation of the RXFP1-signalosome (Halls and Cooper, 2010). At nanomolar concentrations, relaxin stimulation of RXFP1 activates the classical cAMP signalling pathways. RXFP1 can couple to Gα_s to activate AC, and Gα_oB, which inhibits AC activity. The cAMP generated by the combined influence of these two G-protein pathways, ultimately affects CRE-controlled gene transcription. RXFP1 can additionally couple to Gα_i3, which activates a Gβγ-PI3K-PKCζ pathway, resulting in increased AC5 activity. The additional and sustained increases in cAMP generated by this pathway will only affect NFκB-mediated gene transcription. D. Under basal conditions, the β₂-adrenoceptor is also associated with AKAP79, which scaffolds AC5/6 and PKA to the vicinity of the receptor. There is no evidence for cAMP turnover within this complex. E. Following receptor stimulation, the liberation of Gα_s activates AC5/6 to increase cAMP. This results in PKA activation, which phosphorylates the receptor, and can also activate ERK1/2 signalling. The activated receptor can be phosphorylated by GRKs. F. PKA phosphorylation of the receptor C-terminus, results in an uncoupling or signal switching of the β₂-adrenoceptor from Gα_s to Gα_i, leading to receptor desensitization. The Gβγ subunits liberated from Gα_i can also activate ERK1/2 signalling. G. GRK phosphorylation of the receptor C-terminus leads to β-arrestin recruitment and receptor internalization. Recruitment of β-arrestin also allows the scaffolding of Src and PDE4D5 proteins: Src can activate ERK1/2 signalling; whereas PDE4D5 hydrolyses cAMP and inhibits the activity of PKA, preventing signal switching to Gα_i and facilitating desensitization.

Figure 2

The importance of the third intracellular loop and C-terminus of RXFP1 in cAMP signalling. Many important regions for activation of cAMP signalling pathways within the intracellular loops and C-terminal tail of RXFP1 have now been identified. The third intracellular loop and transmembrane domain 6 are important for the activation of cAMP signalling pathways: the C-terminal portion of the third intracellular loop (residues 615–629) is suggested to couple to Gα_s, and mutation of Asp⁶³⁷ induces a constitutive activation of cAMP signalling. The first section of the C-terminal tail regulates the formation of the RXFP1-signalosome: putative helix 8 (residues 673–703) is required for the interaction between RXFP1 and AKAP79 (which scaffolds AC2), and Ser⁷⁰⁴ is absolutely required for the interaction between the C-terminus and β-arrestin 2 (anchoring the regulatory sub-complex). The final 10 residues of the C-terminus (748–757), and specifically Arg⁷⁵² are absolutely required for activation of the Gα_i3–Gβγ–PI3K–PKCζ–AC5 cAMP signalling pathway. The RXFP1 C-terminal tail is not palmitoylated. Symbols above the amino acid residues indicate the relative conservation between RXFP1 and RXFP2: * identical residue, conserved residue and semi-conserved residue.