Phospholipids are needed for the proper formation, stability, and function of the photoactivated rhodopsin-transducin complex.
Heterotrimeric G proteins become activated after they form a catalytically active complex with activated G protein-coupled receptors (GPCRs) and GTP replaces GDP on the G protein alpha-subunit. This transient coupling can be stabilized by nucleotide depletion, resulting in an empty-nucleotide G protein-GPCR complex. Efficient and reproducible formation of conformationally homogeneous GPCR-Gt complexes is a prerequisite for structural studies. Herein, we report isolation conditions that enhance the stability and preserve the activity and proper stoichiometry of productive complexes between the purified prototypical GPCR, rhodopsin (Rho), and the rod cell-specific G protein, transducin (Gt). Binding of purified Gt to photoactivated Rho (Rho*) in n-dodecyl beta-D-maltoside (DDM) examined by gel filtration chromatography was generally modest, and purified complexes provided heterogeneous ratios of protein components, most likely because of excess detergent. Rho*-Gt complex stability and activity were greatly increased by addition of phospholipids such as DOPC, DOPE, and DOPS and asolectin to detergent-containing solutions of these proteins. In contrast, native Rho*-Gt complexes purified directly from light-exposed bovine ROS membranes by sucrose gradient centrifugation exhibited improved stability and the expected 2:1 stoichiometry between Rho* and Gt. These results strongly indicate a lipid requirement for stable complex formation in which the likely oligomeric structure of Rho provides a superior platform for coupling to Gt, and phospholipids likely form a matrix to which Gt can anchor through its myristoyl and farnesyl groups. Our findings also demonstrate that the choice of detergent and purification method is critical for obtaining highly purified, stable, and active complexes with appropriate stoichiometry between GPCRs and G proteins needed for structural studies.
Jastrzebska
Beata
B
Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965, USA.
Goc
Anna
A
Golczak
Marcin
M
Palczewski
Krzysztof
K
D000818:Animals; D002417:Cattle; D019204:GTP-Binding Proteins / Q000378:metabolism; D008027:Light; D008958:Models, Molecular; D010743:Phospholipids / Q000378:metabolism*; D011487:Protein Conformation; D012243:Rhodopsin / Q000737:chemistry / Q000378:metabolism*; D015293:Transducin / Q000737:chemistry / Q000378:metabolism*
D010743:Phospholipids; D012243:Rhodopsin; D019204:GTP-Binding Proteins; D015293:Transducin