title: Facilitatory interplay in alpha 1a and beta 2 adrenoceptor function reveals a non-Gq signaling mode: implications for diversification of intracellular signal transduction.
abstract: Agonist occupied alpha(1)-adrenoceptors (alpha(1)-ARs) engage several signaling pathways, including phosphatidylinositol hydrolysis, calcium mobilization, arachidonic acid release, mitogen-activated protein (MAP) kinase activation, and cAMP accumulation. The natural agonist norepinephrine (NE) activates with variable affinity and intrinsic efficacy all adrenoceptors, and in cells that coexpress alpha(1)- and beta-AR subtypes, such as cardiomyocytes, this leads to coactivation of multiple downstream pathways. This may result in pathway cross-talk with significant consequences to heart physiology and pathologic state. To dissect signaling components involved specifically in alpha(1A)- and beta(2)-AR signal interplay, we have developed a recombinant model system that mimics the levels of receptor expression observed in native cells. We followed intracellular Ca(2+) mobilization to monitor in real time the activation of both G(q) and G(s) pathways. We found that coactivation of alpha(1A)- and beta(2)-AR by the nonselective agonist NE or via a combination of the highly selective alpha(1A)-AR agonist A61603 and the beta-selective agonist isoproterenol led to increases in Ca(2+) influx from the extracellular compartment relative to stimulation with A61603 alone, with no effect on the associated transient release of Ca(2+) from intracellular stores. This effect became more evident upon examination of an alpha(1A)-AR variant exhibiting a partial defect in coupling to G(q), and we attribute it to potentiation of a non G(q)-pathway, uncovered by application of a combination of xestospongin C, an endoplasmic reticulum inositol 1,4,5-triphosphate receptor blocker, and 2-aminoethoxydiphenyl borate, a nonselective storeoperated Ca(2+) entry channel blocker. We also found that stimulation with A61603 of a second alpha(1A)-AR variant entirely unable to signal induced no Ca(2+) unless beta(2)-AR was concomitantly activated. These results may be accounted for by the presence of alpha(1A)/beta(2)-AR heterodimers or alternatively by specific adrenoceptor signal cross-talk resulting in distinct pharmacological behavior. Finally, our findings provide a new conceptual framework to rationalize outcomes from clinical studies targeting alpha- and beta-adrenoceptors.
authors.affiliation: Inflammation Discovery, Roche Palo Alto, Palo Alto, California, USA.
authors.forename: Alicja J
authors.initials: AJ
authors.lastname: Copik
authors.forename: Cynthia
authors.initials: C
authors.lastname: Ma
authors.forename: Alan
authors.initials: A
authors.lastname: Kosaka
authors.forename: Sunil
authors.initials: S
authors.lastname: Sahdeo
authors.forename: Andy
authors.initials: A
authors.lastname: Trane
authors.forename: Hoangdung
authors.initials: H
authors.lastname: Ho
authors.forename: Paul S
authors.initials: PS
authors.lastname: Dietrich
authors.forename: Helen
authors.initials: H
authors.lastname: Yu
authors.forename: Anthony P D W
authors.initials: AP
authors.lastname: Ford
authors.forename: Donald
authors.initials: D
authors.lastname: Button
authors.forename: Marcos E
authors.initials: ME
authors.lastname: Milla
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000502:physiology*; D006333:Heart Failure / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007425:Intracellular Membranes / Q000473:pathology / Q000502:physiology*; D008969:Molecular Sequence Data; D018340:Receptors, Adrenergic, alpha-1 / Q000502:physiology*; D018343:Receptors, Adrenergic, beta-2 / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: C547713:ADRA1A protein, human; D018340:Receptors, Adrenergic, alpha-1; D018343:Receptors, Adrenergic, beta-2; D043802:GTP-Binding Protein alpha Subunits, Gq-G11