A small region in phosducin inhibits G-protein beta gamma -subunit function

Abstract

G‐protein βγ‐subunits (Gβγ) are active transmembrane signalling components. Their function recently has been observed to be regulated by the cytosolic protein phosducin. We show here that a small fragment (amino acids 215–232) contained in the C‐terminus of phosducin is sufficient for high‐affinity interactions with Gβγ. Corresponding peptides not only disrupt Gβγ–Gα interactions, as defined by Gβγ‐stimulated GTPase activity of αo, but also other Gβγ‐mediated functions. The NMR structure of a peptide encompassing this region shows a loop exposing the side chains of Glu223 and Tyr224, and peptides with a substitution of either of these amino acids show a complete loss of activity towards Go. Mutation of this Tyr224 to Ala in full‐length phosducin reduced the functional activity of phosducin to that of phosducin's isolated N‐terminus, indicating the importance of this residue within the short, structurally defined C‐terminal segment. This small peptide derived from phosducin may represent a model of a Gβγ inhibitor, and illustrates the potential of small compounds to affect Gβγ functions.

Effects of two Gβγ-binding proteins – N-terminally truncated phosducin and β-adrenergic receptor kinase C terminus (βARKct) – in heart failure

“…15,17 An alternative explanation would be a specific bARKdirected dominant-negative effect of bARKct, but not of phosducin. Structurally different domains of phosducin seem to mediate binding to Gbg and Gsa.…”

“…15 The Gbgbinding affinities of bARKct (B300 nmol/l), phosducin and an N-terminally truncated variant ('nt-del-phosducin' nt-del-phd) (15 and 33 nmol/l) are comparable. [15][16][17] In contrast to bARKct, however, phosducin and nt-delphosducin do not cause overall resensitization of the badrenergic receptor system owing to simultaneous inhibition of GDP release from Ga subunits. 11,14,18 The structure of phosducin and nt-del-phd are compared schematically in Figure 1.…”

“…Preliminary experiments using various variants of phosducin had shown that truncation of N-terminal sequences resulted in a phosducin with more reproducible Gbg-binding effects in intact cells (Humrich et al, manuscript in preparation). As we had shown before that an N-terminally truncated phosducin (amino acids 64-246) has a Gbg-binding affinity not different from full-length phosducin, 17 we used an Nterminally truncated variant of phosducin starting at the second ATG codon at 156 base pairs ('nt-del-phosducin') for the present study. Overexpression of nt-del-phosducin and bARKct were compared for their effects on contractility and the development of heart failure.…”

Phosphorylation of Phosducin and Phosducin-like Protein by G Protein-coupled Receptor Kinase 2

“…C-terminal hexahistidine-tagged wild-type phosducin (phosducin-His 6 ), a S73A mutant lacking the PKA phosphorylation site, and several N-and C-terminal truncated variants (phosducin-(1-204), phosducin-(64 -245), and phosducin-(138 -245)) were expressed in Escherichia coli strain BL21(DE3)-pLys-S as described previously (27). Wild-type PhLP-(1-218) and its truncated constructs (PhLP-(1-142), PhLP-(1-195), PhLP-(143-218), PhLP-(168 -218)) were expressed as glutathione S-transferase fusion proteins (GST⅐PhLP) as described previously (42).…”

“…The C-terminal region of both phosducin and PhLP have been shown to play a critical role in their interaction with G␤␥ subunits (26,27,30,42). The fact that these regions are also critical for GRK2 phosphorylation suggests that the activity of this kinase may modulate the ability of phosducin to bind G␤␥ dimers.…”