Does coupling to ADP ribosylation factor 6 explain differences between muscarinic and other receptors in interaction with β-adrenoceptor-mediated smooth muscle relaxation?

Numerous studies in airways, ileum, and urinary bladder have demonstrated that relaxation by β-adrenoceptor agonists has lower potency and/or efficacy when contraction was elicited by muscarinic receptor agonists as compared to other G-protein-coupled receptors, KCl, or basal tone, but the molecular mechanisms behind this relative resistance remain unclear. A paper by Huang et al. in this issue demonstrates that NAV2729, an inhibitor of ADP ribosylation factor 6, inhibits contraction of isolated blood vessels elicited by muscarinic receptor agonists, but not by α1-adrenoceptor agonists or KCl. Against this background, we discuss the role of ADP ribosylation factor 6 in cellular responses to G-protein-coupled receptor stimulation. While ADP ribosylation factor 6 apparently is the only promising molecular explanation for the relative resistance of smooth muscle contraction elicited by muscarinic agonists, the existing data are insufficient for a robust conclusion.

Muscarinic receptors are important mediators of smooth muscle contraction in various tissues including airways, gut, and urinary bladder; this largely involves the M 3 subtype but M 2 receptors can also affect smooth muscle tone (Hegde and Eglen 1999). M 3 receptors typically couple to G-proteins of the G q/11 type leading to activation of a phospholipase C (PLC) (Caulfield and Birdsall 1998). Surprisingly, M 3 -mediated smooth muscle contraction is not explained by PLC activation, for instance in the bladder (Frazier et al. 2008), but alternative molecular mechanisms to elicit smooth muscle contraction have not been well-defined.
A paper in this issue of the journal demonstrates that NAV2729, an inhibitor of ADP ribosylation factor 6 (ARF6), inhibits coronary vascular smooth muscle contraction elicited by the muscarinic agonists carbachol and methacholine but not that elicited by agonists at other receptors including α 1 -adrenoceptors, 5-HT, endothelin-1, or prostanoid TP receptors or those elicited by KCl (Huang et al. 2022). The same group had previously shown the existence of ARF6 expression human prostate smooth muscle tissue (Hennenberg et al. 2013) and that NAV2729 inhibited contraction in isolated human prostate strips by α 1 -adrenoceptor agonists, whereas that elicited by a prostanoid TP receptor agonist, endothelin-1 or by KCl, was not inhibited (Yu et al. 2019). Furthermore, NAV2729 inhibited contraction in human prostate smooth muscle cells, and this was also observed in ARF6 knockout cells (Wang et al. 2021). Inhibition of ARF6 by NAV2729 inhibited contraction elicited by α 1adrenoceptor agonists in human prostate (Yu et al. 2019) but not porcine blood vessels (Huang et al. 2022). Interestingly, the inhibition of contraction in human prostate was accompanied by an inhibition of ARF6 in pull-down assays, while ARF6 was not activated by noradrenaline, phenylephrine, or methoxamine (Yu et al. 2019). The selective inhibition of muscarinic receptor responses in the blood vessels (Huang et al. 2022) raises the possibility that coupling to ARF6 may be a mechanism that distinguishes inhibition by β-adrenoceptor agonists of responses to a muscarinic agonist as compared to those elicited by other means.
ARF6 is a small GTP-binding protein that contributes to several cellular processes including G-protein-coupled receptor (GPCR) trafficking, actin organization, and contractile response through diverse downstream component interaction (Fig. 1, Table 1). ARF6 function is modulated by two distinct components. Guanine nucleotide exchange factors (GEFs) mediate the activation of ARF6, whereas GTPaseactivating proteins (GAPs) mediate inhibition. NAV2729 interferes in the formation of ARF6-GEF complex to inhibit the ARF6 activation (Yamauchi et al. 2017).
ARF6 was shown to be a prerequisite component further promoting either clathrin-or caveolin-mediated pathway in agonist-induced endocytosis of several GPCRs such as β-adrenoceptor in adipocytes (Liu et al. 2010) and β 2adrenoceptor in HEK293 cells (Claing et al. 2001;Lawrence et al. 2005;Macia et al. 2012); M 2 muscarinic receptors in HeLa cells (Delaney et al. 2002), in JEG-3 human choriocarcinoma cells (Reiner and Nathanson 2008), and in HEK293 cells (Houndolo et al. 2005); the luteinizing hormone chorionic gonadotropin receptor in HEK 293 cells (Kanamarlapudi et al. 2012); angiotensin type 1 receptor (Houndolo et al. 2005;Cotton et al. 2007); μ-opioid receptor (Rankovic et al. 2009); the vasopressin V 2 receptor; and endothelin type B receptor (Houndolo et al. 2005) in HEK293 cells. Upon agonist stimulation, GPCR endocytosis was found mostly activated via ARF6-dependent pathway with some exceptions which show inhibitor regulatory effect Dashed red arrow, negative regulatory effect; black arrow, positive regulatory effect; GAP, GTPase-activating proteins; GDP, guanosine diphosphate; GEF, guanine nucleotide exchange factors; GTP, guanosine triphosphate; ↑, increase; ↓, decrease; ↔ , no effect  (Delaney et al. 2002;Reiner and Nathanson 2008) and in β 2 -adrenoceptor (Macia et al. 2012) internalization. Furthermore, VIP receptor internalization was not affected by ARF6 depletion (Houndolo et al. 2005). ARF6 involvement in trafficking did not exist for some receptors such as M 4 muscarinic receptor (Reiner and Nathanson 2008) and vasopressin V 2 receptor (Madziva and Birnbaumer 2006). ARF6 requirement in endocytosis was mostly demonstrated in agonist-induced settings, which may not reflect the ARF6 function for basal condition for the same receptor (Cotton et al. 2007). Moreover, ARF6 involvement of muscarinic receptor internalization has mostly been studied with the M 2 subtype because of the well-defined, clathrin-dependent pathway-mediated internalization of M 1 , M 3 , and M 4 receptors (Reiner and Nathanson 2008). Phospholipase D (PLD) is known to be involved in smooth muscle contraction through PKC activation but its contribution in urinary bladder contraction was proposed to be minor (Frazier et al. 2008). ARF6-mediated PLD activation was reported by several researchers in in vivo animal (Le Stunff et al. 2000) and in vitro cultured cell line studies (Mitchell et al. 2003;Johnson et al. 2006;Rankovic et al. 2009;Chakraborti et al. 2017;Charles et al. 2018). In human pulmonary artery smooth muscle cells (HPASMCs), stimulation of prostanoid TP receptor stimulates cytohesin-1 coupling to ARF6 which further leads to PLD2 isoform and subsequent NADPH oxidase activation (Chakraborti et al. 2017). In HPASMCs, involvement of ARF6 in endothelin-1-induced PLD and NADPH oxidase activation was shown by same study group (Chakraborti et al. 2018). ARF6 was shown to be involved in PLD activation in N376D mutant 5-HT 2A -stimulated PLD activation but not in mediated via WT 5-HT 2A stimulation (Johnson et al. 2006). This study also showed the sensitivity of other class A GPCRs which contain DPxxY motif such as purinergic P 2u , thrombin PAR, and gonadotropin-releasing hormone receptor to ARF6 for further PLD activation in COS7 cells (Johnson et al. 2006). In the same cell line, agonist-stimulated M 3 activation induced both PLD1/2 activation through ARF6-mediated pathway, whereas PLD2 activation was found linked to PKC and ARF6 in purinergic P 2U receptor and to only PKC in N376D mutant 5-HT 2a receptor (Mitchell et al. 2003). ARF6 is involved in the promotion of prostate smooth muscle contraction. Inhibition of ARF6 activation by cytohesin (a GEF) inhibitor resulted in reduced noradrenaline, phenylephrine-, thromboxane A 2 -, and endothelin-1-and endothelin-3-induced contraction (Herlemann et al. 2018). In vascular smooth muscle cell, both ARF1 and ARF6 are involved in actin polymerization which subsequently migrate and proliferate but only ARF1 affected contractile responses (Charles et al. 2018). However, in a latter study, ARF6 was found to promote contraction and proliferation in human prostate stromal cells (WPMY-1) (Wang et al. 2021).
ARF6 activation interrupts the recycling of β 2adrenoceptors and lead desensitization of receptor in HEK293 cells (Macia et al. 2012); similarly, it is postulated that ARF6 may involve in α 1 -adrenoceptor desensitization in human prostate tissue (Hennenberg et al. 2013). In contrast, ARF6-mediated endocytosis was suggested beneficial in development of opioid tolerance through preventing receptor desensitization HEK293 cells (Rankovic et al. 2009).
Other effects of ARF6 include the calcium-sensing receptor-mediated plasma membrane ruffling which is required for chemotaxis in HEK cells (Bouschet et al. 2007). In endothelial cells, endothelin ET B receptor stimulation by endothelin 1 results in ARF6 activation which facilitates cell migration via actin reorganization. Moreover, endothelin 1 stimulation did not promote capillary tube formation in ARF6 depleted cells which indicates ARF6 involvement in angiogenesis (Daher et al. 2008). ARF6 also has regulatory effects on metabolic pathway. Depletion of ARF6 resulted in inhibition of isoproterenol-induced lipolysis in 3T3-L1 adipocytes. ARF6 mRNA and protein level was found higher in WAT tissue of ob/ob mice compared to obesity resistance mice (Liu et al. 2010). In 3T3-L1 adipocytes, endothelin ET A receptor-mediated lipolysis was found to be dependent downstream ARF6-ERK1/2 signaling (Davies et al. 2014). Additionally, endothelin 1 stimulated GLUT4 translocation through heterotrimetric G q protein signaling pathway activated by ARF6 in 3T3-L1 adipocytes (Bose et al. 2001;  Lawrence and Birnbaum 2001) and specifically G α11 isoform of G αq family. Taken together, the above data demonstrate that ARF6 is involved in cellular processes and is activated by cellular effects stimulated by various GPCR, including smooth muscle contraction. Within a given tissue, the involvement of ARF6 in pathways leading to smooth muscle contraction, e.g., in arteries or prostate, appears to be preferential for some GPCR (muscarinic receptors and α 1 -adrenoceptors, respectively) over others apparently coupling to the same G-proteins. Some of these data are in line with the hypothesis that coupling to ARF6 may explain the relative resistance of smooth muscle contraction elicited by muscarinic receptors as compared to other GPCR or receptor-independent contraction such as receptor desensitization to relaxation by β-adrenoceptor agonists. However, not all data support this hypothesis. Thus, the role of ARF6 in this phenomenon cannot be considered proven, but it remains as a reasonable molecular candidate to explain the resistance of muscarinic receptor-mediated smooth muscle contraction against relaxation. Further studies at the molecular level are required to further explore this, specifically studies in which the role of agonists at various GPCR is compared quantitatively.