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Expression of functional G protein‐coupled receptors in photoreceptors of transgenic Xenopus laevis
Author(s) -
Zhang Li,
Salom David,
He Jianhua,
Okun Alex,
Ballesteros Juan,
Palczewski Krzysztof,
Li Ning
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a919-b
Subject(s) - rhodopsin , g protein coupled receptor , xenopus , biology , receptor , microbiology and biotechnology , g protein , transmembrane protein , transmembrane domain , signal transduction , biochemistry , gene , retinal
G protein‐coupled receptors (GPCRs) represent the largest superfamily of transmembrane signaling proteins; however, the only known GPCR crystal structure is that of rhodopsin. This disparity reflects the difficulty in generating purified GPCR samples of sufficient quantity and quality. Rhodopsin, the light receptor of retinal rod neurons, is produced in large amounts of homogenous quality in the vertebrate retina. We used transgenic Xenopus laevis to convert these retina rod cells into bioreactors to successfully produce 20 model GPCRs. The receptors accumulated in rod outer segments and were homogenously glycosylated. Ligand and [ 35 S]GTPγ S binding assays of the 5HT 1A and EDG 1 GPCRs confirmed that they were properly folded and functional. 5HT 1A R was highly purified by taking advantage of the rhodopsin C‐terminal immunoaffinity tag common to all GPCR constructs. This strategy can be extended to other animal model systems with similar rhodopsin biosynthesis machinery, which has been demonstrated by the homogeneous expression of several GPCRs in transgenic mouse retinas. This expression approach could become a general method for the production of large numbers of GPCRs and other membrane proteins for pharmacological and structural studies. This research was supported by SBIR Grant R43MH068919‐1 from the National Institute of Mental Health and SBIR Contract N43‐CM‐37011‐1 from the National Cancer Institute.