Constitutively Active Mutants of the α1a- and the α1b-Adrenergic Receptor Subtypes Reveal Coupling to Different Signaling Pathways and Physiological Responses in Rat Cardiac Myocytes

Charlene Mcwhinney,Dean Wenham,Sujata Kanwal,Vivian Kalman,Carl Hansen,Janet D Robishaw

doi:10.1074/jbc.275.3.2087

Charlene Mcwhinney, Dean Wenham + Show 4 more

Open Access

https://doi.org/10.1074/jbc.275.3.2087

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Abstract

Activation of alpha(1)-adrenergic receptors influences both the contractile activity and the growth potential of cardiac myocytes. However, the signaling pathways linking activation of specific alpha(1)-adrenergic receptor (AR) subtypes to these physiological responses remain controversial. In the present study, a molecular approach was used to identify conclusively the signaling pathways activated in response to the individual alpha(1A)- and alpha(1B)-AR subtypes in cardiac myocytes. For this purpose, a mutant alpha(1a)-AR subtype (alpha(1a)-S(290/293)-AR) was constructed based on analogy to the previously described constitutively active mutant alpha(1b)-AR subtype (alpha(1b)-S(288-294)-AR). The mutant alpha(1a)-S(290/293)-AR subtype displayed constitutive activity based on four criteria. To introduce the constitutively active alpha(1)-AR subtypes into cardiac myocytes, recombinant Sindbis viruses encoding either the alpha(1a)-S(290/293)-AR or alpha(1b)-S(288-294)-AR subtype were used to infect the whole cell population with >90% efficiency, thereby allowing the biochemical activities of the various signaling pathways to be measured. When expressed at comparable levels, the alpha(1a)-S(290/293)-AR subtype exhibited a significantly elevated basal level as well as agonist-stimulated level of inositol phosphate accumulation, coincident with activation of atrial natriuretic factor-luciferase gene expression. By contrast, the alpha(1b)-S(288-294)-AR subtype displayed a markedly increased serum response element-luciferase gene expression but no activation of atrial natriuretic factor-luciferase gene expression. Taken together, this study provides the first molecular evidence for coupling of the alpha(1a)-AR and the alpha(1b)-AR subtypes to different signaling pathways in cardiac myocytes.

Highlights

¶ To whom correspondence should be addressed: Henry Hood Research Program, Pennsylvania State College of Medicine, Weis Center for Research, 100 North Academy Ave., Danville, PA 17822
We demonstrated the ability of a recombinant Sindbis virus to infect greater than 90% of the cardiac myocytes, as measured by positive ␤-galactosidase staining
Construction and Characterization of the Constitutively Active ␣1a-S290/293-adrenergic receptor (AR)—To address whether constitutively active mutants of the ␣1a-AR and the ␣1b-AR subtypes could be used to probe their subtype-specific coupling to signaling pathways, a constitutively active mutant of the ␣1a-AR needed to be constructed

Summary

Introduction

¶ To whom correspondence should be addressed: Henry Hood Research Program, Pennsylvania State College of Medicine, Weis Center for Research, 100 North Academy Ave., Danville, PA 17822. Differential Coupling of Active ␣1-AR Subtypes the limitations of a pharmacologic approach, we sought to develop a molecular approach that could be used to identify conclusively which signaling pathways, and which functional responses, are activated in response to the individual ␣1A- and ␣1B-AR subtypes in cardiac myocytes. To this end, we constructed a constitutively active mutant of the ␣1a-AR subtype by analogy to a previously described constitutively active mutant of the ␣1b-AR subtype [9]. Following introduction of the individual constitutively active ␣1-AR subtypes into the normal cellular context of cardiac myocytes in which the wild type receptor subtypes are expressed, we determined which signaling pathways are activated in response to each mutant receptor subtype in the absence of agonist

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