Molecular Mechanism of Transmembrane Signal 
Transduction by G Protein-coupled Receptors 
Thomas p. Sakmar, M.D. — Assistant Investigator 
Dr. Sakmar is also Assistant Professor at the Rockefeller University. He received his A.B. degree in 
chemistry and his M.D. degree from the University of Chicago. He completed a medical residency at 
Massachusetts General Hospital, Boston, and conducted postdoctoral research in the laboratory 
of H. Gobind Khorana at the Massachusetts Institute of Technology. 
IN our laboratory the vertebrate visual proteins 
rhodopsin and transducin serve as a model sys- 
tem for structure-function studies on the molecu- 
lar mechanism of transmembrane signaling. 
These visual proteins are members of a superfam- 
ily of related G proteins (guanine-binding regula- 
tory proteins) and G protein-coupled receptors. 
Light-activated rhodopsin catalyzes guanine nu- 
cleotide exchange by transducin, v^^hich ulti- 
mately leads to a change in membrane cation 
conductance and a neural signal. Our approach is 
to reconstitute heterologously expressed rho- 
dopsin and transducin in defined in vitro systems 
and to use biochemical and biophysical methods 
to probe site-directed mutants. 
Our current interests include structure- 
function relationships in rhodopsin. For exam- 
ple, we are studying the ground state structure of 
the receptor, the interactions between specific 
amino acid residues and the 1 l-c?s-retinal chro- 
mophore that control spectral properties and 
photochemistry, the mechanism by which a pho- 
tochemical signal is transmitted from the core of 
the receptor to the surface, and the specific do- 
mains on the cytoplasmic surface that bind and 
activate transducin. 
We employ a multifaceted approach, including 
the use of a variety of complementary spectro- 
scopic techniques. For example, the structure 
and environment of the retinal chromophore in 
rhodopsin and its photointermediates can be stud- 
ied with resonance Raman spectroscopy. In a con- 
tinuation of collaborative work with Steven Lin 
and Richard Mathies, we obtained microprobe 
resonance Raman spectra of solutions containing 
only microgram quantities of mutant pigments. 
The results confirmed and supplemented our 
earlier observations concerning the role of a spe- 
cific carboxylate group in rhodopsin that acts to 
stabilize the positive charge of the protonated 
Schiff base chromophore linkage. A model of the 
chromophore binding pocket of rhodopsin was 
proposed and is being used to direct further stud- 
ies into the mechanism of wavelength regulation 
by visual pigments. 
We are also interested in identifying specific 
domains of rhodopsin and transducin involved in 
binding and activation. Flash photolysis studies 
of site-directed rhodopsin mutants had previ- 
ously shown that at least the second and third 
cytoplasmic loops of rhodopsin are involved in 
activation of bound transducin. Some cytoplas- 
mic mutations prevent transducin binding as 
well. Recently we have developed a spectrofluo- 
rimetric method designed to allow simultaneous 
illumination and measurements of rhodopsin- 
transducin interactions by intrinsic fluorescence. 
Rhodopsin-catalyzed binding of GTP, or a GTP 
analogue to transducin, results in a large increase 
in its intrinsic fluorescence. Mixtures of transdu- 
cin and rhodopsin can be assayed by this method 
to determine the kinetic rate constants of their 
interaction and to evaluate the specific effects of 
mutations. A series of site-directed mutants of 
rhodopsin with alterations in their cytoplasmic 
domains have been studied. The results may be 
relevant to other seven-transmembrane helix re- 
ceptors that couple to G proteins and play roles 
in cellular physiology, growth, development, 
and differentiation in the nervous system. 
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