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  • Typically upon chronic agonist exposure GPCRs undergo desens


    Typically, upon chronic agonist exposure GPCRs undergo desensitization and internalization resulting in a loss of receptor responsiveness over time (Drake et al., 2006, Kelly et al., 2008). However, not all GPCR systems conform to this model of acute agonist-mediated regulation. Some receptors have been reported not to internalize and/or desensitize (Callander et al., 2009, Jensen et al., 2013, Willars et al., 1999). Furthermore it has recently been demonstrated that when conventional desensitization mechanisms do not exert a major regulatory influence, sustained receptor responses can be maintained with persistent agonist–receptor interactions. Functionally, the consequences of this mechanism have been defined as sustained receptor activation even after removal of excess agonist (Calebiro et al., 2009, Ferrandon et al., 2009, Haskell-Luevano et al., 1996, Mullershausen et al., 2009). Whilst some evidence for GPR119 desensitization has been previously reported (Kumar et al., 2014, Lauffer et al., 2009, Zhang et al., 2014), a comprehensive characterization of desensitization of GPR119 cAMP signalling has not yet been conducted. This key gap in our understanding of GPR119 biology may be especially pertinent to observations of tachyphylaxis and lack of efficacy of GPR119 agonists in the clinic (Kang, 2013). In the present study we have investigated the chronic effects of the endogenous GPR119 agonist oleoylethanolamide as well as the synthetic agonists AR-231,453, GSK-1292263, MBX-2982, AZ1, AZ2, and AZ3, as described previously (Brocklehurst et al., 2011, Scott et al., 2012, Semple et al., 2008) and shown in Fig. 1. Using a recombinant beta amyloid system we find that desensitization is not a major regulatory influence. Additionally, some agonists demonstrate sustained wash-resistant receptor activation, and we provide experimental evidence to support a role for slow dissociation kinetics or membrane deposition in mediating these responses.
    Materials and methods
    Discussion Classic understanding of GPCR signalling is described by a gradual loss of receptor responsiveness after prolonged agonist exposure caused by desensitization and internalization via β‐arrestin‐dependent mechanisms (Drake et al., 2006, Kelly et al., 2008). However, we find that in our test system, desensitization is not a major regulatory influence on GPR119 function as little/no loss of cAMP response is observed with chronic agonist exposure. Direct reports of GPR119 desensitization in the literature are limited, although a recent study demonstrated desensitization of calcium responses by novel and existing GPR119 agonists using re-stimulation experiments (Zhang et al., 2014). This need not be inconsistent with our findings as pathway-specific or agonist-specific desensitization mechanisms potentially play. Additionally, the kinetics of the calcium response and levels of receptor reserve may also contribute to the observed differences in desensitization profile between these systems. A second recent report suggested that both AR-231,453 and oleoylethanolamide evoke complete desensitization of GPR119 cAMP responses (Kumar et al., 2014). However, in these experiments the investigators did not appear to wash out excess ligand before challenging with a second stimulus, which may preclude the observation of re-stimulation responses and therefore confound the observation of true desensitization. To our knowledge, this is the first direct and comprehensive investigation of agonist-mediated desensitization of GPR119 cAMP responses. A number of other GPCR systems have also been reported to show no loss of responsiveness upon chronic stimulation with agonist (Calebiro et al., 2009, Callander et al., 2009, Ferrandon et al., 2009, Jensen et al., 2013, Mullershausen et al., 2009, Willars et al., 1999). We cannot rule out desensitization at very high agonist concentrations, as this was observed in some cases with supramaximal stimulation in an assay-specific manner. Lauffer et al. (2009) speculated that bell-shaped concentration–responses curves to GPR119 agonists indicate receptor desensitization at high concentrations. Indeed, we also observe bell-shaped curves for some agonists. It is plausible that supramaximal concentrations of agonists evoke desensitization by either non-conventional mechanisms or due to low affinity interactions with β-arrestin-coupled conformations of the receptor. Nevertheless, robust receptor activation is still observed after chronic exposure to pharmacologically relevant concentrations of our test agonists.