Enhanced beta2-adrenergic receptor (beta2AR) signaling by adeno-associated viral (AAV)-mediated gene transfer.
BACKGROUND
Beta2-adrenergic receptors (beta2AR) play important regulatory roles in a variety of cells and organ systems and are important therapeutic targets in the treatment of airway and cardiovascular disease. Prolonged use of beta-agonists results in tolerance secondary to receptor down-regulation resulting in reduced therapeutic efficiency. The purpose of this work is to evaluate the signaling capabilities of the beta2AR expressed by a recombinant adeno-associated viral (AAV) vector that also included an enhanced green fluorescent protein (EGFP) gene (AAV-beta2AR/EGFP).


RESULTS
By epifluorescence microscopy, approximately 40% of infected HEK 293 cells demonstrated EGFP expression. beta2AR density measured with [3H]dihydroalprenolol ([3H]DHA) increased either 13- or 77-fold in infected cells compared to mock infected controls depending on the culture conditions used. The [3H]DHA binding was to a single receptor population with a dissociation constant of 0.42 nM, as would be expected for wild-type beta2AR. Agonist competition assays with [3H]DHA showed the following rank order of potency: isoproterenol>epinephrine> norepinephrine, consistent with beta2AR interaction. Isoproterenol-stimulated cyclic AMP levels were 5-fold higher in infected cells compared to controls (314 +/- 43 vs. 63.4 +/- 9.6 nmol/dish; n = 3). Receptor trafficking demonstrated surface expression of beta2AR with vehicle treatment and internalization following isoproterenol treatment.


CONCLUSIONS
We conclude that HEK 293 cells infected with AAV-beta2AR/EGFP effectively express beta2AR and that increased expression of these receptors results in enhanced beta2AR signaling. This method of gene transfer may provide an important means to enhance function in in vivo systems.
Jones
Stacie M
SM
Department of Pediatrics, University of Arkansas for Medical Sciences, Arkansas Children's Hospital, Little Rock, Arkansas 72202, USA. JonesStacieM@uams.edu
Hiller
F Charles
FC
Jacobi
Sandie E
SE
Foreman
Susan K
SK
Pittman
Laura M
LM
Cornett
Lawrence E
LE
D000256:Adenoviridae / Q000235:genetics; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
D008164:Luminescent Proteins; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP