Heterologous expression of the cloned guinea pig α2A, α2B, and α2C adrenoceptor subtypes: Radioligand binding and functional coupling to a cAMP-responsive reporter gene

Samuel P.S. Svensson, Thomas J. Bailey, Amy C. Porter, Jeremy G. Richman, John W. Regan

Research output: Contribution to journalArticlepeer-review

23 Scopus citations


Functional studies have shown that 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4,5-tetrahydro-3- benzazepine (SKF 104078) has very low affinity for prejunctional α2-adrenoceptors (α2-AR) in the guinea pig atrium. In this study, we have cloned guinea pig homologues of the human α2-C10, α2-C2 and α2-C4 AR subtypes and have studied them in isolation by heterologous expression in cultured mammalian cells. Oligonucleotide primers, designed from conserved areas of the human α2-ARs were used in a polymerase chain reaction (PCR) with template cDNA synthesized from guinea pig atrial mRNA. Three PCR products were obtained that shared identity with the three human α2-AR subtypes. A guinea pig (gp) genomic library was screened with a cDNA clone encoding a portion of the gp-α2A, and genes containing the complete coding sequences of the guinea pig α2A, α2B, and α2C AR subtypes were obtained. These guinea pig genes were subcloned into a eukaryotic expression plasmid and were expressed transiently in COS-7 cells. The binding of the α2-selective antagonist [3H]MK-912 to membranes prepared from these cells was specific and of high affinity with Kd values of 810 pM for gp-α2A, 2700 pM for gp-α2B and 110 pM for gp-α2C. Competition for the binding of [3H]MK-912 by SKF 104078 indicated that it was of moderately high affinity (∼100 nM) but that it was not selective for any of the guinea pig α2-AR subtypes. Co-expression of guinea pig α2-AR subtypes with a cyclicAMP-responsive chloramphenicol acetyltransferase (CAT) reporter gene resulted in agonist-dependent modulation of CAT activity. For the gp-α2A, a biphasic response was obtained with low concentrations of noradrenaline (NE) decreasing forskolin-stimulated CAT activity and high concentrations causing a reversal. For the gp-α2B, NE produced mostly potentiation of forskolin-stimulated activity, and for the gp-α2C, NE caused mainly inhibition. Overall, the pharmacology of the cloned guinea pig α2-AR subtypes was in agreement with data obtained for the native guinea pig receptors and was functionally similar to that of the cloned human α2-AR subtypes.

Original languageEnglish (US)
Pages (from-to)291-300
Number of pages10
JournalBiochemical Pharmacology
Issue number3
StatePublished - Feb 9 1996


  • Adenylyl cyclase
  • G-protein coupled
  • Pharmacology
  • Reporter gene

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology


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