Signal transfer from GPCRs to G proteins: role of the G alpha N-terminal region in rhodopsin-transducin coupling.
Catalysis of nucleotide exchange in heterotrimeric G proteins (Galphabetagamma) is a key step in cellular signal transduction mediated by G protein-coupled receptors. The Galpha N terminus with its helical stretch is thought to be crucial for G protein/activated receptor (R(*)) interaction. The N-terminal fatty acylation of Galpha is important for membrane targeting of G proteins. By applying biophysical techniques to the rhodopsin/transducin model system, we studied the effect of N-terminal truncations in Galpha. In Galphabetagamma, lack of the fatty acid and Galpha truncations up to 33 amino acids had little effect on R(*) binding and R(*)-catalyzed nucleotide exchange, implying that this region is not mandatory for R(*)/Galphabetagamma interaction. However, when the other hydrophobic modification of Galphabetagamma, the Ggamma C-terminal farnesyl moiety, is lacking, R(*) interaction requires the fatty acylated Galpha N terminus. This suggests that the two hydrophobic extensions can replace each other in the interaction of Galphabetagamma with R(*). We propose that in native Galphabetagamma, these two terminal regions are functionally redundant and form a microdomain that serves both to anchor the G protein to the membrane and to establish an initial docking complex with R(*). Accordingly, we find that the native fatty acylated Galpha is competent to interact with R(*) even in the absence of Gbetagamma, whereas nonacylated Galpha requires Gbetagamma for interaction. Experiments with N-terminally truncated Galpha subunits suggest that in the second step of the catalytic process, the receptor binds to the alphaN/beta1-loop region of Galpha to reduce nucleotide affinity and to make the Galpha C terminus available for subsequent interaction with R(*).
Herrmann
Rolf
R
Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Schumannstrasse 20/21, D-10098 Berlin, Germany.
Heck
Martin
M
Henklein
Peter
P
Hofmann
Klaus Peter
KP
Ernst
Oliver P
OP
D000595:Amino Acid Sequence; D000818:Animals; D044385:GTP-Binding Protein alpha Subunits / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000502:physiology*; D012243:Rhodopsin / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D015293:Transducin / Q000378:metabolism*
D044385:GTP-Binding Protein alpha Subunits; D043562:Receptors, G-Protein-Coupled; D012243:Rhodopsin; D015293:Transducin