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Background and Purpose: β-Adrenoceptor agonists relieve airflow obstruction by activating β 2 -adrenoceptors, which are G protein-coupled receptors (GPCRs) expressed on human airway smooth muscle (HASM) cells. The currently available β-adrenoceptor agonists are balanced agonists, however, and signal through both the stimulatory G protein (G s )- and β-arrestin-mediated pathways. While G s signalling is beneficial and promotes HASM relaxation, β-arrestin activation is associated with reduced G s efficacy. In this context, biased ligands that selectively promote β 2 -adrenoceptor coupling to G s signalling represent a promising strategy to treat asthma. Here, we examined several β-adrenoceptor agonists to identify G s -biased ligands devoid of β-arrestin-mediated effects., Experimental Approach: G s -biased ligands for the β 2 -adrenoceptor were identified by high-throughput screening and then evaluated for G s interaction, G i interaction, cAMP production, β-arrestin interaction, GPCR kinase (GRK) phosphorylation of the receptor, receptor trafficking, ERK activation, and functional desensitization of the β 2 -adrenoceptor., Key Results: We identified ractopamine, dobutamine, and higenamine as G s -biased agonists that activate the G s /cAMP pathway upon β 2 -adrenoceptor stimulation while showing minimal G i or β-arrestin interaction. Furthermore, these compounds did not induce any receptor trafficking and had reduced GRK5-mediated phosphorylation of the β 2 -adrenoceptor. Finally, we observed minimal physiological desensitization of the β 2 -adrenoceptor in primary HASM cells upon treatment with biased agonists., Conclusion and Implications: Our work demonstrates that G s -biased signalling through the β 2 -adrenoceptor may prove to be an effective strategy to promote HASM relaxation in the treatment of asthma. Such biased compounds may also be useful in identifying the molecular mechanisms that determine biased signalling and in design of safer drugs., (© 2022 British Pharmacological Society.)

Upon activation, many G protein-coupled receptors (GPCRs) internalize by clathrin-mediated endocytosis and are subsequently sorted to undergo recycling or lysosomal degradation. Here we observe that sorting can take place much earlier than previously thought, by entry of different GPCRs into distinct populations of clathrin-coated pit (CCP). These distinct populations were revealed by analysis of two purinergic GPCRs, P2Y(1) and P2Y(12), which enter two populations of CCPs in a mutually exclusive manner. The mechanisms underlying early GPCR sorting involve differential kinase-dependent processes because internalization of P2Y(12) is mediated by GPCR kinases (GRKs) and arrestin, whereas P2Y(1) internalization is GRK- and arrestin-independent but requires protein kinase C. Importantly, the beta(2) adrenoceptor which also internalizes in a GRK-dependent manner also traffics exclusively to P2Y(12)-containing CCPs. Our data therefore reveal distinct populations of CCPs that sort GPCR cargo at the plasma membrane using different kinase-dependent mechanisms.

In human hypertension we have recently identified an increase in lymphocyte G-protein receptor kinase-2 (GRK-2) protein expression, the key protein regulating the interaction between G-protein-coupled receptors and activation of adenylyl cyclase. However, it was not known whether this increase in GRK-2 protein expression was attributable to regulation at the level of translation. Furthermore, the relationship between extent of GRK-2 expression, receptor activation of adenylyl cyclase, and blood pressure was unclear. We therefore studied lymphocytes from 7 young subjects with borderline hypertension and 14 young normotensive subjects. Immunodetectable GRK-2 protein expression in lymphocytes from subjects with hypertension was increased (155%+/-7% of normotensive subjects; P < .05). In addition, GRK-2 protein expression was positively correlated with blood pressure (r = 0.53; P = .013) and inversely correlated with beta-adrenergic-mediated adenylyl cyclase activity (r = -0.54, P = .012). However, lymphocyte GRK-2 messenger ribonucleic acid (mRNA) content was not altered (110%+/-13% of that observed in normotensive control subjects). Increased GRK-2 protein expression may be an important factor in the impairment of beta-adrenergic-mediated vasodilation, characteristic of the hypertensive state.

1. G protein-coupled receptor kinases (GRKs) are thought to be important in mediating the agonist-induced phosphorylation and consequent desensitization of G protein-coupled receptor (GPCR) responses. We have previously shown that stable expression of a dominant negative mutant G protein-coupled receptor kinase 2 (GRK2) construct in NG108-15 mouse neuroblastoma x rat glioma cells suppresses the agonist-induced desensitization of A2A and A2B adenosine receptor-stimulated adenylyl cyclase activity (Mundell et al., 1997). To further determine the role of GRK2 in agonist-induced desensitization of these adenosine receptors, we stably overexpressed wild type GRK2 in NG108-15 cells. 2. In homogenates prepared from cells overexpressing GRK2, the acute stimulation of adenylyl cyclase by activation of A2A and A2B adenosine receptors was markedly reduced, but could be reversed by pretreating the cells with AD (adenosine deaminase), to remove extracellular adenosine from the medium. On the other hand, acute stimulation of adenylyl cyclase by secretin, iloprost, NaF and forskolin was the same in GRK2 overexpressing cells and plasmid-transfected control cells. 3. Cells overexpressing GRK2 were more sensitive to adenosine receptor agonist-induced desensitization than plasmid-transfected control cells. This effect was selective since the agonist sensitivity of desensitization for secretin and IP-prostanoid receptor-stimulated adenylyl cyclase activity was not affected by GRK2 overexpression. 4. These results further implicate GRK2 as the likely mechanism by which A2 adenosine receptors undergo short-term desensitization in NG108-15 cells, and indicate that even when overexpressed, GRK2 retains its substrate specificity for native receptors in intact cells. Furthermore, the susceptibility of GPCRs to desensitization appears to depend on the level of GRK expression, such that in cells that express high levels of GRK2, low agonist concentrations may be sufficient to trigger GRK-mediated desensitization.