Membrane organization and function of the serotonin(1A) receptor

Abstract

(1) The serotonin(1A) receptor is a G-protein coupled receptor involved in several cognitive, behavioral, and developmental functions. It binds the neurotransmitter serotonin and signals across the membrane through its interactions with heterotrimeric G-proteins. (2) Lipid-protein interactions in membranes play an important role in the assembly, stability, and function of membrane proteins. The role of membrane environment in serotonin(1A) receptor function is beginning to be addressed by exploring the consequences of lipid manipulations on the ligand binding and G-protein coupling of serotonin(1A) receptors, the ability to functionally solubilize the serotonin(1A) receptor, and the factors influencing the membrane organization of the serotonin(1A) receptor. (3) Recent developments involving the application of detergent-based and detergent-free approaches to understand the membrane organization of the serotonin(1A) receptor under conditions of ligand activation and modulation of membrane lipid content, with an emphasis on membrane cholesterol, are described.

Fig. 1

Chemical structures of ligands that bind to the serotonin_1A receptor

Fig. 2

A schematic representation of the membrane embedded human serotonin_1A receptor showing its predicted topological and other structural features. The membrane is shown as a bilayer of two leaflets of lipids. The amino acids in the receptor sequence are shown as circles and are marked after every 50 residues for convenience. Seven transmembrane regions, each composed of 20–26 amino acids, are depicted as α-helices. There are three potential sites of N-linked glycosylation on the amino terminus (depicted as branching trees). A putative disulfide bond between Cys-109 and Cys-187 is shown. Transmembrane (TM) domains contain residues (which are marked) that are important for ligand binding. Putative palmitoylation sites are Cys-417 and/or Cys-420. Light gray circles represent contact sites for G-proteins. Black circles represent sites for protein kinase mediated phosphorylation. Adapted from Pucadyil et al. (2005a)

Fig. 3

Detergent insolubility of the serotonin_1A receptor fused to EYFP. Cells expressing serotonin_1A-EYFP receptors are shown (A) before and (B) after treatment with cold Triton X-100 (0.05%, w/v) for 10 min. The images represent combined mid-plane confocal sections of the same group of cells before and after detergent extraction. The scale bar represents 10 μm. Reproduced from Kalipatnapu and Chattopadhyay (2004b)

Fig. 4

Isolation and analysis of membrane fractions from bovine hippocampal membranes using a detergent-free method to isolate membrane domains. Panel A shows the typical pattern of isolation of light, heavy, and extra heavy membrane fractions from hippocampal membranes on a sucrose density gradient using the detergent-free method of Luria et al. (2002). The membrane fraction at 10–22.5% sucrose interface is designated as ‘light’, at 22.5–35% sucrose interface as ‘heavy’, and a faintly visible fraction at 35–40% sucrose interface as ‘extra heavy’. The light and heavy membrane fractions have been shown to be derived primarily from the plasma membrane, whereas the extra heavy fraction is shown to be mainly from intracellular components (Monneron and d’Alayer ; Luria et al. 2002). Comparison of ligand binding to serotonin_1A receptors from the light and heavy membrane fractions isolated using a detergent-free method from native hippocampal membranes is shown in panel B. The white bars represent the binding of the agonist [³H]8-OH-DPAT and the shaded bars that of the antagonist [³H]p-MPPF. Data obtained from radioligand binding assays have been represented as a percentage of the total recovered ligand binding obtained from the light and heavy membrane fractions in order to appreciate the distribution of serotonin_1A receptors among the light and heavy membrane fractions. The data points represent means ± SD of duplicate points from three independent experiments. Adapted and modified from Kalipatnapu and Chattopadhyay (2007)