Heterologous expression of the adenosine A1 receptor in transgenic mouse retina.
Traditional cell-based systems used to express integral membrane receptors have yet to produce protein samples of sufficient quality for structural study. Herein we report an in vivo method that harnesses the photoreceptor system of the retina to heterologously express G protein-coupled receptors in a biochemically homogeneous and pharmacologically functional conformation. As an example we show that the adenosine A1 receptor, when placed under the influence of the mouse opsin promoter and rhodopsin rod outer segment targeting sequence, localized to the photoreceptor cells of transgenic retina. The resulting receptor protein was uniformly glycosylated and pharmacologically well behaved. By comparison, we demonstrated in a control experiment that opsin, when expressed in the liver, had a complex pattern of glycosylation. Upon solubilization, the retinal adenosine A1 receptor retained binding characteristics similar to its starting material. This expression method may prove generally useful for generating high-quality G protein-coupled receptors for structural studies.
Li
Ning
N
Novasite Pharmaceuticals Inc., San Diego, California 92121, USA.
Salom
David
D
Zhang
Li
L
Harris
Tim
T
Ballesteros
Juan A
JA
Golczak
Marcin
M
Jastrzebska
Beata
B
Palczewski
Krzysztof
K
Kurahara
Carole
C
Juan
Todd
T
Jordan
Steven
S
Salon
John A
JA
D000818:Animals; D002460:Cell Line; D005818:Genetic Engineering; D006801:Humans; D008024:Ligands; D051379:Mice; D008822:Mice, Transgenic; D020419:Photoreceptor Cells, Vertebrate / Q000737:chemistry; D043682:Receptor, Adenosine A1 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012160:Retina / Q000166:cytology / Q000378:metabolism*
D008024:Ligands; D043682:Receptor, Adenosine A1