Functional incorporation of G protein‐coupled receptors into reconstituted HDL

Abstract

High density lipoproteins (HDL) are lipid-protein complexes that are involved in the reverse transport of cholesterol. When HDL is first formed in the body, it is disc shaped, with a diameter of 10–12 nm. It is comprised of a dimer of amphipathic, helically structured apolipoprotein A-I (apoA-I) wrapped around a phospholipid bilayer. The HDL complex can be reconstituted in vitro by combining purified apoA-I with phospholipid micelles. We are developing a system whereby we incorporate G protein-coupled receptors (GPCRs) into the phospholipid bilayer of rHDL. We have succeeded in functionally incorporating the two prototypic GPCRs into rHDL: the β2 adrenergic receptor (β2AR) and rhodopsin. β2AR incorporated into reconstituted HDL (rHDL) binds [3H]dihydroalprenolol with a Kd~0.5 nM, representing a ten-fold higher affinity than observed in detergent micelles and more consistent with the Kd values found with β2AR in cellular membranes. In addition, the receptor exhibits both high affinity agonist binding and agonist-stimulated guanine nucleotide exchange. Rhodpsin incorporated into rHDL comprised of unsaturated lipids converts to the active metarhodopsin II conformation with properties consistent with that seen in rod outer segment membranes upon light activation. In the case of both receptors, alterations in the lipid composition have dramatic consequences on the efficiency of receptor incorporation and function, consistent with similar studies using phospholipid vesicles or lamellar preparations. With this approach both the disc size and phospholipid composition may be controlled giving this powerful technique the potential to be applied to both the structural and functional characterization of membrane proteins in general. Supported by NIH RO1 GM-068603