Abstract
G-protein coupled receptors (GPCRs) are integral membrane proteins involved in a wide variety of biological processes in eukaryotic cells, and are targeted by a large fraction of marketed drugs. GPCR kinases (GRKs) play important roles in feedback regulation of GPCRs, such as of β-adrenergic receptors in the heart, where GRK2 and GRK5 are the major isoforms expressed. Membrane targeting is essential for GRK function in cells. Whereas GRK2 is recruited to the membrane by heterotrimeric Gβγ subunits, the mechanism of membrane binding by GRK5 is not fully understood. It has been proposed that GRK5 is constitutively associated with membranes through elements located at its N-terminus, its C-terminus, or both. The membrane orientation of GRK5 is also a matter of speculation. In this work, we combined sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) to help determine the membrane orientation of GRK5 and a C-terminally truncated mutant (GRK51-531) on membrane lipid bilayers. It was found that GRK5 and GRK51-531 adopt a similar orientation on model cell membranes in the presence of PIP2 that is similar to that predicted for GRK2 in prior studies. Mutation of the N-terminal membrane binding site of GRK5 did not eliminate membrane binding, but prevented observation of this discrete orientation. The C-terminus of GRK5 does not have substantial impact on either membrane binding or orientation in this model system. Thus, the C-terminus of GRK5 may drive membrane binding in cells via interactions with other proteins at the plasma membrane or bind in an unstructured manner to negatively charged membranes.
Highlights
The duration of signaling by most G-protein coupled receptors (GPCRs) is regulated by the activity of GPCR kinases (GRKs), which phosphorylate the third cytoplasmic loop or C-terminal tails of activated receptors and thereby initiate their uncoupling from heterotrimeric G proteins and subsequent downregulation [1]
We began by collecting sum frequency generation (SFG) amide I signal on 9:1 POPC:POPG lipid bilayers that were previously used for orientation analysis of Gβ1γ2, GRK2-Gβ1γ2 and Gαiβ1γ2 [5,25]
This result either indicates that GRK5 does not bind to these bilayers, or that when bound to the bilayer it exhibits a random distribution of orientations such that SFG signals from its α–helical components cancel out
Summary
The duration of signaling by most G-protein coupled receptors (GPCRs) is regulated by the activity of GPCR kinases (GRKs), which phosphorylate the third cytoplasmic loop or C-terminal tails of activated receptors and thereby initiate their uncoupling from heterotrimeric G proteins and subsequent downregulation [1]. Palmitoylation does not occur in GRK5 and is not required for membrane binding or the function of this enzyme [11,12,13,14] It is not clear if GRK4 subfamily members associate with membranes in an orientation similar to that predicted for GRK2, or whether a specific orientation is induced only in the presence of certain anionic phospholipids and/or by activated receptors. Because of their homology and the fact that they phosphorylate many GPCRs with similar catalytic efficiency, it is anticipated that the membrane-bound orientations of all GRKs are similar
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