Genetic Polymorphisms Affect Mouse and Human Trace Amine-Associated Receptor 1 Function

Abstract

Methamphetamine (MA) and neurotransmitter precursors and metabolites such as tyramine, octopamine, and β-phenethylamine stimulate the G protein-coupled trace amine-associated receptor 1 (TAAR1). TAAR1 has been implicated in human conditions including obesity, schizophrenia, depression, fibromyalgia, migraine, and addiction. Additionally TAAR1 is expressed on lymphocytes and astrocytes involved in inflammation and response to infection. In brain, TAAR1 stimulation reduces synaptic dopamine availability and alters glutamatergic function. TAAR1 is also expressed at low levels in heart, and may regulate cardiovascular tone. Taar1 knockout mice orally self-administer more MA than wild type and are insensitive to its aversive effects. DBA/2J (D2) mice express a non-synonymous single nucleotide polymorphism (SNP) in Taar1 that does not respond to MA, and D2 mice are predisposed to high MA intake, compared to C57BL/6 (B6) mice. Here we demonstrate that endogenous agonists stimulate the recombinant B6 mouse TAAR1, but do not activate the D2 mouse receptor. Progeny of the B6XD2 (BxD) family of recombinant inbred (RI) strains have been used to characterize the genetic etiology of diseases, but contrary to expectations, BXDs derived 30-40 years ago express only the functional B6 Taar1 allele whereas some more recently derived BXD RI strains express the D2 allele. Data indicate that the D2 mutation arose subsequent to derivation of the original RIs. Finally, we demonstrate that SNPs in human TAAR1 alter its function, resulting in expressed, but functional, sub-functional and non-functional receptors. Our findings are important for identifying a predisposition to human diseases, as well as for developing personalized treatment options.

Fig 1. B6 vs D2 SNP density on proximal chromosome 10 (from 5 kb– 33.8 Mb).

SNPs between B6 and D2 were counted in 5kb bins. D10Mit28 and D10Mit3 are the original PCR markers used in the BXD RI genotyping spanning the mTaar1 region [–31]. The green shading indicates the 7 Mb region in which mTaar1 has the only D2 missense SNP.

Fig 2. β-PEA and tyramine stimulate recombinant B6 but not D2 mTAAR1.

The endogenous ligands, β-PEA (A) and tyramine (B) elicited a dose-dependent response in mammalian cells expressing recombinant B6-like, but not the D2-like (P77T) mTAAR1. Dose-response curves for the recombinant B6-like receptor were shifted to the right by EPPTB (10 μM). β-PEA EC₅₀ = 0.547± 0.03 μM; Tyramine EC₅₀ = 0.940 ± 0.12 μM. LSD has no effect on either receptor (C).

Fig 3. Schematic transmembrane topology of hTAAR 1.

(Adapted from [35].) Amino acid residues incorporated in the transmembrane domains are shaded in gray, residues putatively involved in ligand binding are colored red. N-linked glycosylation at N 10 and N 17, as well as the disulfide bridge linking C 96 and C 182, is indicated according to the annotation in Swiss Prot entry TAR1_HUMAN. Known non-synonymous SNPs in the hTAAR1 that may produce changes in receptor function are boxed in blue (as determined from NCBI SNP Database). Indicated is the amino acid change induced by the SNP as: reference amino acid, residue number, change amino acid (Term = Termination). Further details, including the SNP nucleotide change are provided in the text.

Fig 4. β-PEA and MA stimulate hTAAR1, and hTAAR1 SNPs reduce or eliminate receptor function.

A. β-PEA and MA increased cAMP production in CHO cells expressing the hTAAR1. There was a significant dose × agonist interaction (F (18, 56) = 15.70, p<0.0001. *p < 0.01 for comparison between the indicated group and the untransfected control. ⁺p < 0.01 for comparison between PEA and MA groups. B. β-PEA dose-response curves in untransfected CHO cells and cells expressing the hTAAR1 variants. EPPTB (10 μM) caused a rightward shift in the β-PEA dose-response curve. There was a significant dose × receptor variant interaction (F (42,147) = 3.189; p<0.0001). * p<0.05 comparing C182Y and untransfected control cells; ** p<0.0001. ns = non-significant. Data shown are means ± SEMs from at least three independent experiments, each conducted with duplicate determinations. C. Confocal images of receptor expression in transfected cells.