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Introduction

The neurotensin receptor (NTSR1) belongs to the superfamily of proteins known as G protein-coupled receptors (GPCRs) and responds to the 13 amino acid hormone neurotensin (NTS). There are currently around 800 G protein-coupled receptors that have been identified and are thought to be responsible for roughly 80% of signal transduction across the cell membrane.^[1] These receptors are involved in a vast array of physiological processes within the body that range from interactions with dopamine to effects on secretion of bile in the intestines.^{[2] [3]} Due to the vast array of functions that these proteins serve and their high abundance within the body, these proteins have become a major site of drug targets in medicine making a deeper, more in depth understanding of these proteins very important. ^[4] There are currently no NTRS1 structures of the inactive state, so there is no way to determine the conformational change of the binding pocket caused by the binding of NTS. ^[5]

Neurotensin

is a 13-amino acid peptide originally isolated from bovine hypothalamus. It fulfills the role of both a neurotransmitter and a neuromodulator in the nervous system and a hormone in the periphery. NTS is a neuromodulator of dopamine transmission and of anterior pituitary hormone secretion. It is also a paracrine and endocrine modulator in the periphery of the digestive tract and cardiovascular system. Finally, NTS serves as a growth factor for many normal and cancerous cell types.^[6]

Only the C-terminal tail of NTS, amino acids 8-13, were resolved in the crystal structure.

Structure

Overall Structure

Like other G protein-coupled receptors, the neurotensin receptor is composed of 3 distinct regions. An extracellular binding site where neurotensin binds and causes a conformational change of the protein, a region containing that transduce the signal from the extracellular side of the cell membrane to the intracellular side, and an intracellular region that, when activated by a conformational change in the protein, activates a G protein associated with this receptor. There are currently no crystal structures of the inactive form of the neurotensin receptor available. Without a representation of the inactive form, the conformational changes caused by agonist binding are still not completely known.

Neurotensin Binding Site

Binding of NTS to the binding site is enriched by between the positive NTS arginine side chains and the electronegative pocket. In addition, the C-terminus forms a with R328. Only three out of eight hydrogen bonds are made between the side chains of NTS and the receptor. Most of the interactions are van der Waals interactions. The binding pocket is partially capped by a β-hairpin loop at the proximal end of the receptor protein's N-terminus.^[5]

Hydrophobic Stacking

A major player in the transduction of the extracellular signal to the intracellular G protein is the that links the bound hormone with the hydrophobic core of the neurotensin receptor. The carboxylate of L13 forms a hydrogen bond network with R327, R328, and Y324. The Y324, in turn, is brought into an orientation to make the formation of a network between F358, W321, A157, and F317 possible.^[7] The conformational changes caused by this stacking allows for the signal to be moved from the extracellular binding site through the transmembrane helices of the receptor to the intracellular region activating the G protein.

Sodium Binding Pocket

Conserved across all class A GPCRs, a is seen in the middle of TM2 helix. The ion is coordinated with a highly conserved D^2.50 and four other contacts with oxygen atoms. Some of these oxygen atoms are sourced from water molecules. In order for G-protein activation, a with T^3.39, S^7.46 ,N^7.49 of the NPxxY motif, prevents the coordination of a Na⁺. ^[7][8]

Allosteric Effects

Sodium ions are a negative allosteric inhibitor to the binding of the neurotensin agonist to the binding site on the neurotensin receptor. D113 of the highly conserved D/RY motif and N365 of the highly conserved NPxxY motif form a substantial hydrogen bonding network with T156 and S362.^[7] This hydrogen bonding network prevents the incorporation of the sodium ion by collapsing upon itself and therefor filling the sodium binding pocket. W321 also works to inhibit the incorporation of the sodium ion by capping off the sodium binding pocket to not allow sodium to enter from the top. W321 uses van der Walls interactions with other amino acids in the binding pocket to place it in the conformation necessary to complete this task.

Clinical Relevance

NTSR1 is commonly expressed in various invasive cancer cell lines. It is prevalent in the colon cancer adenocarcinoma, but is not found in adult colon cell types. NTSR1 is also found in aggressive prostate cancer cells, but not epithelial prostate cells. In prostate cancer cells, binding of the NTS results in mitogen-activated protein kinase (PKB), phosphoinositide-3 kinase (PI-3K), epidermal growth factor receptor (EGFR), SRC, and STAT5 phosphorylation. These all result in increased DNA sythesis, cell proliferation, and survival.

http://cancerres.aacrjournals.org/content/71/21/6817.long

http://cancerres.aacrjournals.org/content/69/16/6539.full.pdf+html