Contribution of cyclic nucleotides to CGRP receptor functions
In the vasculature, CGRP receptor activation causes relaxation of VSM (Brain and Grant, 2004; Meens et al., 2009; 2010). This has been linked to Gα-mediated pathways like activation of AC and opening of KATP channels (Brain and Grant, 2004). In addition, CGRP receptor activation promotes dissociation of established ET-1/ETA complexes and thus terminates ET-1-induced signalling in rat mesenteric arteries, i.e. CGRP-/ETA receptor cross-talk (Meens et al., 2010). The CGRP-induced dissociation of ET-1 from ETA receptors is (i) not mimicked by vasodilators that act through activation of AC, release of NO or opening of KATP channels (Meens et al., 2010), and (ii) can also be observed in a variety of other rat arteries and is sufficiently widespread to affect systemic effects of ET-1 in intact rats (Meens et al., 2011). This indicates that the CGRP receptor-induced dissociation of ET-1 from ETA receptors is widely distributed over the arterial tree and suggests that it is not mediated by Gαs. In addition, these observations indicate that the relaxation induced by CGRP receptor activation and the dissociation of ET-1 from its receptor may be initiated by different molecular mechanisms. In this study, the presence of IBMX did not increase, and the presence of ODQ did not reduce, responses to CGRP in rat isolated MRA, although inhibitors of PDE or soluble guanylyl cyclase changed the responses to K+, SNP and ISO as expected (Hilgers and De Mey, 2009; Meens et al., 2009; 2010). Thus, arterial relaxing and anti-ET-1 effects of CGRP receptor activation seem to be independent of cyclic nucleotides. We therefore investigated the role of Gβγ subunits of GTP binding regulatory proteins (Gβγ) in the functions of the CGRP receptor.
Contribution of Gβγ to CGRP receptor functions in isolated arteries
Inhibition of Gβγ subunits using two recently described Gβγ inhibitors (Bonacci et al., 2006; Lehmann et al., 2008) did not reduce but rather increased binding of [125I]-CGRP. The exact molecular mechanism by which gallein increased agonist binding remains to be established, but there are several candidates. (i) CLR- and RAMP1-homodimers exist in the plasmalemma (Heroux et al., 2007). Gβγ may be involved in stabilization of these homodimers. In such a scenario, inhibition of Gβγ would result in increased formation of RAMP1/CLR heterodimers which would increase the Bmax for [125I]-CGRP. (ii) Subsets of the total CGRP receptor population can exist in either a high or a low-affinity state, depending on pre-coupling to Gαβγ (Maton et al., 1988; Wimalawansa and MacIntyre, 1988; Chatterjee and Fisher, 1991; van Rossum et al., 1993; Schindler and Doods, 2002; Gales et al., 2006). Therefore, as gallein displayed its effects in the presence of GTPγS (Figure 3), Gβγ can bind directly to the parathyroid hormone 1 receptor (Mahon et al., 2006), a related class B 7TM receptor, and mathematical models predict a negative effect of Gβγ on receptor ligand binding affinity (Onaran et al., 1993). Gβγ may directly retain CGRP receptors in a low-affinity state that we could not have observed in our experiments due to a limited amount of radioligand. In addition to increasing [125I]-CGRP binding, gallein increased cAMP production induced by CGRP and ISO in cultured VSMCs, in line with recent findings by others in cardiomyocytes (Casey et al., 2010). However, perhaps due to the abundant expression of PDEs, this did not seem to affect the vasorelaxing effects of ISO or CGRP and the anti-endothelinergic effects of CGRP receptor activation. Therefore, we investigated the contribution of Gβγ to CGRP receptor-induced effects in isolated arteries. Both the vasorelaxing and the anti-ET-1 effects of exogenous CGRP were selectively and concentration-dependently inhibited by the Gβγ inhibitors gallein and M119. Thus, similar to other 7TM receptors (Smrcka, 2008), CGRP receptors cause intracellular signalling and relaxation of VSM via Gβγ. Previously, Gβγ have been shown to activate or inhibit various effector proteins such as phospholipases, AC, K+ channels, G-protein receptor kinases and PI3K (Sunahara et al., 1996; Schneider et al., 1997; Vanhaesebroeck et al., 1997; Bonacci et al., 2006). The effector protein(s) involved in the arterial effects of CGRP remain to be directly demonstrated, but at least AC and KATP do not seem to be involved (Meens et al., 2009; 2010 and Figure 2 of this study). PI3K and phospholipases are pobably not involved because (i) the presence of wortmannin did not affect the vascular effects of CGRP, and (ii) activation of these proteins has mostly been linked to intracellular pathways that enhance, rather than inhibit, vasoconstriction (Somlyo and Somlyo, 2003; Yin and Janmey, 2003). Thus, the exact intracellular mechanism involved in the cross-talk between CGRP and ETA receptors remains to be unraveled, but was at least found to involve Gβγ. Cross-talk between various 7TM receptors has been proposed by mathematical modelling (Quitterer and Lohse, 1999; Flaherty et al., 2008; Cervantes et al., 2010; Tubio et al., 2010). It can involve ‘G-protein hijacking’ (Tubio et al., 2010), cross-talk via arrestin (Cervantes et al., 2010) and formation of receptor heterodimers or oligomers (Prezeau et al., 2010). In addition, Gβγ involved in receptor cross-talk have been identified before they are, for example, implicated in stimulating cross-talk between Gα(i)- and Gα(q)-coupled receptors (Quitterer and Lohse, 1999). However, many, if not all, of these molecular mechanisms suggested to be involved in 7TM receptor cross-talk to date can potentially also affect the contractile apparatus of VSMC, and our results do not provide information regarding possible Gβγ effector proteins. Therefore, at present we cannot address the amount of convergence between the signalling pathways involved in the relaxing and anti-endothelinergic effects of CGRP receptor stimulation. In a previous study, we used fluorescently labelled ET-1 to directly monitor the dissociation of ET-1/ETA receptor complexes in intact isolated arteries (Meens et al., 2010). Unfortunately, the dye-like properties of gallein, M119 and fluorescein prevent the inclusion of these compounds in such molecular imaging experiments.
In conclusion, our data indicate that CGRP receptor activation causes cAMP production but the relaxation of rat MRA induced by activation of this receptor involves Gβγ and is not dependent on cAMP. Moreover, in rat MRA, CGRP receptors terminate the effects of ET-1 via Gβγ, which also reduces CGRP binding to CGRP receptors (Figure 10). In the future, these findings may lead to new drugs for both CGRP- and ET-1-related diseases. Orthosteric Gβγ-biased (Zheng et al., 2010) CGRP receptor agonists could be used to terminate the effects of ET-1 without causing side effects due to Gαs-mediated effects. Small molecular weight Gβγ inhibitors, which have already shown beneficial effects in animal studies focusing on inflammation and heart failure (Lehmann et al., 2008; Casey et al., 2010), could be used for treatment of diseases characterized by either an excess of Gβγ-mediated effects of CGRP or a defect in Gαs-mediated signalling by the peptide.