Quantification of functional selectivity at the human α(1A)-adrenoceptor.

Although G protein-coupled receptors are often categorized in terms of their primary coupling to a given type of Gα protein subunit, it is now well established that many show promiscuous coupling and activate multiple signaling pathways. Furthermore, some agonists selectively activate signaling pathways by promoting interaction between distinct receptor conformational states and particular Gα subunits or alternative signaling proteins. We have tested the capacity of agonists to stimulate Ca(2+) release, cAMP accumulation, and changes in extracellular acidification rate (ECAR) at the human α(1A)-adrenoceptor. Signaling bias factors were determined by novel application of an operational model of agonism and compared with the reference endogenous agonist norepinephrine; values significantly different from 1.0 indicated an agonist that promoted receptor conformations distinct from that favored by norepinephrine. Oxymetazoline was a full agonist for ECAR and a partial agonist for Ca(2+) release (bias factor 8.2) but failed to stimulate cAMP production. Phenylephrine showed substantial bias toward ECAR versus Ca(2+) release or cAMP accumulation (bias factors 21 and 33, respectively) but did not display bias between Ca(2+) and cAMP pathways. Cirazoline and N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide (A61603) displayed bias toward cAMP relative to Ca(2+) release (bias factors of 7.4 and 8.6). It is noteworthy that epinephrine, a second endogenous adrenoceptor agonist, did not display bias relative to norepinephrine. Our finding that phenylephrine displayed significant signaling bias, despite being highly similar in structure to epinephrine, indicates that subtle differences in agonist-receptor interaction can affect conformational changes in cytoplasmic domains and thereby modulate the repertoire of effector proteins that are activated.

Factors influencing biased agonism in recombinant cells expressing the human α1A -adrenoceptor.

BACKGROUND AND PURPOSE: Agonists acting at GPCRs promote biased signalling via Gα or Gßγ subunits, GPCR kinases and ß-arrestins. Since the demonstration of biased agonism has implications for drug discovery, it is essential to consider confounding factors contributing to bias. We have examined bias at human α1A -adrenoceptors stably expressed at low levels in CHO-K1 cells, identifying off-target effects at endogenous receptors that contribute to ERK1/2 phosphorylation in response to the agonist oxymetazoline. EXPERIMENTAL APPROACH: Intracellular Ca2+ mobilization was monitored in a Flexstation® using Fluo 4-AM. The accumulation of cAMP and ERK1/2 phosphorylation were measured using AlphaScreen® proximity assays, and mRNA expression was measured by RT-qPCR. Ligand bias was determined using the operational model of agonism. KEY RESULTS: Noradrenaline, phenylephrine, methoxamine and A61603 increased Ca2+ mobilization, cAMP accumulation and ERK1/2 phosphorylation. However, oxymetazoline showed low efficacy for Ca+2 mobilization, no effect on cAMP generation and high efficacy for ERK1/2 phosphorylation. The apparent functional selectivity of oxymetazoline towards ERK1/2 was related to off-target effects at 5-HT1B receptors endogenously expressed in CHO-K1 cells. Phenylephrine and methoxamine showed genuine bias towards ERK1/2 phosphorylation compared to Ca2+ and cAMP pathways, whereas A61603 displayed bias towards cAMP accumulation compared to ERK1/2 phosphorylation. CONCLUSION AND IMPLICATIONS: We have shown that while adrenergic agonists display bias at human α1A -adrenoceptors, the marked bias of oxymetazoline for ERK1/2 phosphorylation originates from off-target effects. Commonly used cell lines express a repertoire of endogenous GPCRs that may confound studies on biased agonism at recombinant receptors.

Optimizing the connectivity in disulfide-rich peptides: alpha-conotoxin SII as a case study.

We describe a strategy for the efficient, unambiguous assignment of disulfide connectivities in alpha-conotoxin SII, of which approximately 30% of its mass is cysteine, as an example of a generalizable technique for investigation of cysteine-rich peptides. alpha-Conotoxin SII was shown to possess 3-8, 2-18, and 4-14 disulfide bond connectivity. Sequential disulfide bond connectivity analysis was performed by partial reduction with Tris(2-carboxyethyl)phosphine and real-time mass monitoring by direct-infusion electrospray mass spectrometry (ESMS). This method achieved high yields of the differentially reduced disulfide bonded intermediates and economic use of reduced peptide. Intermediates were alkylated with either N-phenylmaleimide or 4-vinylpyridine. The resulting alkyl products were assigned by ESMS and their alkyl positions sequentially identified via conventional Edman degradation. The methodology described allows a more efficient, rapid, and reliable assignment of disulfide bond connectivity in synthetic and native cysteine-rich peptides.

The Janus faces of adrenoceptors: factors controlling the coupling of adrenoceptors to multiple signal transduction pathways.

1. The adrenoceptors (AR) are an important subfamily of rhodopsin-like G-protein-coupled receptors that couple to an increasingly large number of signalling mechanisms. Two important factors that determine the pathways that are used are the C-terminal region of the receptor and the agonist used to activate the receptor. 2. Studies of splice variants of the mouse beta3-AR showed that the C-terminus is a factor controlling the signalling characteristics. Although these receptors differ only at the C-terminus, the beta3b-AR coupled to both Gs and Gi, whereas the beta3a-AR coupled solely to Gs. 3. Examination of four splice variants of the human alpha1A-AR showed that all were able to couple to pertussis toxin-sensitive G-proteins, even though they have radically different C-terminal regions. 4. Comparison of the effects of the beta3-AR ligands CL316243 and SR59230A showed that both can activate the mouse beta3-AR but that SR59230A uses pathways other than cAMP accumulation in 3T3-F442A cells. 5. Examination of a series of alpha1-AR agonists for their ability to activate a number of signalling pathways revealed that A61603 acted as a full agonist in all assays, whereas oxymetazoline was unable to cause cAMP accumulation, suggesting agonist-selective signalling at the human alpha1A-AR.