The identification and functional implications of human-specific "fixed" amino acid substitutions in the glutamate receptor family.

BACKGROUND: The glutamate receptors (GluRs) play a vital role in the mediation of excitatory synaptic transmission in the central nervous system. To clarify the evolutionary dynamics and mechanisms of the GluR genes in the lineage leading to humans, we determined the complete sequences of the coding regions and splice sites of 26 chimpanzee GluR genes.
RESULTS: We found that all of the reading frames and splice sites of these genes reported in humans were completely conserved in chimpanzees, suggesting that there were no gross structural changes in humans after their divergence from the human-chimpanzee common ancestor. We observed low KA/KS ratios in both humans and chimpanzees, and we found no evidence of accelerated evolution. We identified 30 human-specific "fixed" amino acid substitutions in the GluR genes by analyzing 80 human samples of seven different populations worldwide. Grantham's distance analysis showed that GRIN2C and GRIN3A are the most and the second most diverged GluR genes between humans and chimpanzees. However, most of the substitutions are non-radical and are not clustered in any particular region. Protein motif analysis assigned 11 out of these 30 substitutions to functional regions. Two out of these 11 substitutions, D71G in GRIN3A and R727H in GRIN3B, caused differences in the functional assignments of these genes between humans and other apes.
CONCLUSION: We conclude that the GluR genes did not undergo drastic changes such as accelerated evolution in the human lineage after the divergence of chimpanzees. However, there remains a possibility that two human-specific "fixed" amino acid substitutions, D71G in GRIN3A and R727H in GRIN3B, are related to human-specific brain function.

Background

Glutamate is the most abundant fast-excitatory neurotransmitter in the central nervous system (CNS) and glutamate receptors (GluRs) play a vital role in the mediation of excitatory synaptic transmission. Because of their roles in neurotransmission and synaptic plasticity, GluRs are thought to be key molecules in cognitive functions such as learning and memory (reviewed in [1-3]). Based on their structural and functional characteristics, GluRs are classified into two major groups: ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs) (Reviewed in [3]). Vertebrate iGluRs are pharmacologically classified into four subgroups by their ligand selectivity: NMDA, AMPA, kainate, and delta. While iGluRs directly regulate the ion flux across the cell membrane as ion channels, mGluRs are involved in a variety of intracellular signaling pathways by activating phospholipase C and/or suppressing adenlylate cyclase and subsequently mediating excitatory neurotransmission and synaptic plasticity by affecting iGluR activities.

Recent studies have reported that the genetic variations in GluRs are associated with multiple neurobehavioral phenotypes in humans including addictions, anxiety/dysphoria disorders, schizophrenia, and epilepsy [e.g., [4-12]]. These observations suggest that genetic variations in GluRs cause brain dysfunctions in humans. A study of the evolutionary genetic changes in the GluR genes would provide us with insights into the molecular basis of human-specific brain and nervous system functions.

The evolutionary changes that occurred in the GluR genes in the lineage leading to humans are poorly understood, mainly because of the limited availability of relevant information in public databases. To overcome this problem, we determined the complete coding sequences of 26 GluR genes in chimpanzees (Pan troglodytes) and conducted a comparative genomic analysis of all GluR genes. We examined whether positive selection plays a role in the evolution of the GluR gene family after the divergence of humans and chimpanzees and investigated human-specific "fixed" nonsynonymous substitutions in the GluR genes that might be associated with human-specific brain function.

Results

We determined the complete nucleotide sequences of the coding exons for 21 chimpanzee glutamate receptor (GluR) genes: GRIA3, GRIA4, GRID1, GRID2, GRIK1, GRIK2, GRIK3, GRIK4, GRIK5, GRIN1, GRIN2A, GRIN2C, GRIN2D, GRIN3A, GRIN3B, GRM1, GRM2, GRM4, GRM6, GRM7, and GRM8. The sequences of five additional chimpanzee GluR genes, GRIA1, GRIA2, GRIN2B, GRM3, and GRM5 were obtained from the UCSC Genome Database (version panTro1) [13]. Successful alignments of the entire coding regions and splice sites of the human and chimpanzee GluR genes indicated no gross structural differences such as protein truncation between humans and chimpanzees.

The pairwise nucleotide divergence

We calculated the pairwise nucleotide divergence in total and synonymous sites between humans and chimpanzees for all of the genes encoding NMDA, AMPA, kainate, delta, and metabotropic glutamate receptors (Table 1). The divergence for the entire set of GluR genes both at total and synonymous sites was 0.00461 and 0.01257, respectively, which is significantly lower than the genome-wide average values, which are 0.0059 and 0.0177 (more than 2 SD below the mean in a normal distribution, [14]). Indeed, except for the NMDA type, the divergence for each type was significantly lower than the genome-wide average at total and synonymous sites (more than 2 SD below the mean).

Table 1

The pairwise nucleotide divergence per site at total and synonymous sites between humans and chimpanzees

Type	Total sites	Synonymous sites
AMPA	0.00242 ± 0.00053	0.00792 ± 0.00167
Delta	0.00432 ± 0.00076	0.01341 ± 0.00292
Kainate	0.00429 ± 0.00047	0.01288 ± 0.00166
NMDA	0.00594 ± 0.00045	0.01477 ± 0.00141
mGluR	0.00444 ± 0.00041	0.01186 ± 0.00122
All GluRs	0.00461 ± 0.00019	0.01257 ± 0.00056

The AMPA type genes showed the lowest divergence of the GluR types at both total and synonymous sites (0.00242 ± 0.00053 and 0.00792 ± 0.00167 at total and synonymous sites, respectively). The divergence of the four individual AMPA genes ranged from 0.00075 to 0.00303 at total sites and from 0.00056 to 0.00103 at synonymous sites. Therefore, the AMPA type genes showed lower divergence than the other GluR types. The divergence for the NMDA type genes was the highest of all of the GluR types at both total and synonymous sites (0.00594 ± 0.00045 and 0.01477 ± 0.00141). This observed higher divergence can be attributed to two NMDA type GluR genes, GRIN3A and GRIN3B. The GRIN3B and GRIN3A genes are the most and the second most diverged GluR genes between humans and chimpanzees at both types of site (0.01351 and 0.00965 at total sites and 0.03023 and 0.02844 at synonymous sites). When we excluded GRIN3A and GRIN3B from this analysis, the NMDA type genes were found to have similar divergences to the other gene types at total and synonymous sites (0.00425 ± 0.00040 and 0.01154 ± 0.00135).

Lineage-specific KA/KS ratios and selection tests

Intrigued by the possible functional implications of the GluR genes in the evolutionary process of the human lineage, we examined whether positive selection acted on the GluR genes in humans and chimpanzees. At first, using macaque sequences as an outgroup, we calculated the human and chimpanzee lineage-specific nonsynonymous and synonymous substitution rates (Kand K, respectively) and their ratios (K/K, Table 2). Notably, the K/Kratios for the GluR genes were less than one in both the human and chimpanzee lineages, although we could not calculate the ratios for 11 human and 11 chimpanzee GluR genes due to the absence of nonsynonymous substitutions. Excluding the genes with no substitutions, the K/Kratios ranged from 0.0004 to 0.417 and 0.0005 to 0.242 in human and chimpanzee lineages, respectively. The K/Kratios for 24 human GluR genes and all of the chimpanzee GluR genes are smaller than the species-specific genome-wide mean values (0.259 and 0.245 in human and chimpanzee, respectively [15]). These results indicate that the functional constraint on the GluR is relatively strong.

Table 2

The nonsynonymous and synonymous rates and their ratio in the human and chimpanzee lineages

	Human			Chimpanzee
gene	KA	KS	KA/KS	KA	KS	KA/KS
GRIA1	0	0.0051	0	0	0.0053	0
GRIA2	0	0.0041	0	0	0.0069	0
GRIA3	0	0.0012	0	0.0005	0	0
GRIA4	0.0006	0.0059	0.0933	0	0.0023	0
GRID1	0.0009	0.0093	0.0947	0	0.007	0
GRID2	0	0.0109	0	0.0005	0.0032	0.1495
GRIK1	0	0.0024	0	0	0.0106	0
GRIK2	0	0.0028	0	0	0.0043	0
GRIK3	0.0005	0.0189	0.0247	0.0005	0.0197	0.0236
GRIK4	0.0004	0.0139	0.0323	0	0.0064	0
GRIK5	0.0013	0.0055	0.2313	0.0004	0.0041	0.1025
GRIN1	0	0.0159	0	0	0.0051	0
GRIN2A	0.0009	0.0066	0.1396	0.0009	0.0075	0.1244
GRIN2B	0	0.0078	0	0.0003	0.0058	0.0518
GRIN2C	0.0023	0.013	0.1745	0.0015	0.0129	0.1157
GRIN2D	0	0.013	0	0	0.005	0
GRIN3A	0.0026	0.0087	0.2976	0.0017	0.015	0.1131
GRIN3B	0.0017	0.0447	0.0388	0	0	0
GRM1	0.0004	0.0082	0.0466	0.0004	0.0042	0.0901
GRM2	0.0005	0.0108	0.049	0.0005	0.0074	0.0709
GRM3	0.0005	0.0013	0.417	0.0011	0.0044	0.2422
GRM4	0	0.0109	0	0.0005	0.0069	0.0663
GRM5	0	0.0031	0	0	0.0057	0
GRM6	0.0025	0.0224	0.1123	0.0016	0.0159	0.1038
GRM7	0.0005	0.0105	0.0514	0.0011	0.0071	0.1521
GRM8	0.0005	0.0039	0.1327	0.001	0.0055	0.1902

K, K, and K/Kindicate nonsynonymous, synonymous, and the nonsynonymous/synonymous substitution rate (per site), respectively.

Two maximum likelihood ratio tests (implemented in PAML [16]) were employed to examine the K/Kfor the GluR genes in the human lineage and to evaluate the accelerated selection for the GluR genes in humans and chimpanzees. First, we compared the K/Kratios in the human lineage vs. background (chimpanzee and macaque) lineages. We observed that the human-specific K/Kratio is significantly different from the background ratio in GRM7 according to test B outlined in [17] (Additional file 1). Taking the K/Kvalue into account, this result implies that the human K/Kratio is significantly lower than that of the background in GRM7. We then applied the improved branch-site model [18] to examine whether positive selection acted on the GluR genes in humans and chimpanzees, but could not detect any significant accelerated selection in either human or chimpanzee lineages (Additional file 2).

Identification of human-specific "fixed" nonsynonymous mutations

To evaluate the functional changes of the GluR genes in the human lineage, we searched for human-specific "fixed" nonsynonymous substitutions. First, we carried out a pairwise comparison between the human and chimpanzee orthologs of 26 GluR genes. We found a total of 80 nonsynonymous substitutions including four indels (insertion/deletion) (Table 3). Out of the 80 substitutions, two substitutions were excluded from further analysis due to discrepancies among the UCSC reference and our chimpanzee sequences; these two substitutions are possibly polymorphic within chimpanzees. Second, we sequenced the remaining 78 substitution/indel sites in five additional apes: the bonobo, gorilla, orangutan, siamang, and crab-eating macaque. We regarded human alleles that were not shared with any of the great apes to be "human-specific". We pooled substitutions that were found specifically either in chimpanzees or in bonobos as "chimpanzee-specific" substitutions, because these mutations must have occurred in the chimpanzee and bonobo lineages after the divergence of the human lineage. Out of the 78 human-chimpanzee substitution sites, we identified 37 human-specific (35 substitutions and 2 indels) and 31 chimpanzee-specific substitutions/indels (29 substitutions and 2 indels). The remaining 10 are recurrent substitutions in the primate lineages. To determine whether these substitutions/indels are "fixed" or "polymorphic" in human populations, we sequenced 80 human samples representing seven different populations around the world for the 37 human-specific substitution/indel sites. Table 3 shows the 30 "fixed" (28 substitutions and 2 indels) and 7 "polymorphic" substitutions/indels that we confirmed in the human populations. The 30 human-specific "fixed" substitutions are potentially responsible for human-specific functions.

Table 3

Comparison of human-chimpanzee substitutions among primates

ID#	Gene	Amino acid (Hum-Chimp)	Nucleotide (Hum-Chimp)	Hum	Chimp	Bon	Gor	Ora	Gib	Mac #1	Mac #2	Specificity1	Fixed in Humans	Notes
1	GRIN2A	S906N	AGC-AAC	G	A	A	A	A	A	A	A	Human	Fixed
2	GRIN2A	A1006V	GCG-GTG	C	T	T	T	T	T	T	T	Human	Fixed
3	GRIN2A	H1080P	CAC-CCC	A	C	A	A	A	A	A	A	Chimpanzee
4	GRIN2A	F1158L	TTC-TTG	G	C	C	G	G	G	G	G	Chimpanzee
5	GRIN2A	H1173Q	CAT-CAA	A	T	T	A	A	A	A	A	Chimpanzee
6	GRIN2A	M1221L	ATG-CTG	A	C	C	C	C	C	C	C	Human	Fixed
7	GRIN2B	N1294T	AAC-ACC	A	C	C	A	A	A	A	A	Chimpanzee
8	GRIN2C	P23L	CCG-CTG	CCG	ATG	CTG	CCG	CTG	CGG	CCG	CCG	N.A.
9	GRIN2C	T71N	ACC-AAC	C	A	A	A	C	C	A	A	N.A.
10	GRIN2C	H89R	CAC-CGC	A	G	G	G	G	G	G	G	Human	Fixed
11	GRIN2C	D100G	GAC-GGC	A	G	G	G	G	G	G	G	Human	Fixed
12	GRIN2C	A596S	GCT-TCT	G	T	T	T	"T"	T	T	T	Human	Fixed
13	GRIN2C	S851T	TCC-ACC	T	A	A	T	T	T	T	T	Chimpanzee
14	GRIN2C	Q898R	CAG-CGG	A	A/G	-	A	A	A	A	A	Chimpanzee
15	GRIN2C	S933P	TCC-CCC	T	C	-	-	C	C	"C"	"C"	Human	Fixed
16	GRIN2C	G1144S	GGC-AGC	G	A	A	G*	G	G	"G"	"G"	Chimpanzee		*AGG
17	GRIN2C	R1221C	CGT-TGT	C	T	T	-	"T"	T	"T"	"T"	Human	Fixed
18	GRIN3A	S30G	AGC-GGC	A	G	G	-	"G"	G	G	G	Human	Fixed
19	GRIN3A	D71G	GAC-GGC	A	G	G	-	"G"	G	G	G	Human	Fixed
20	GRIN3A	P93L	CCG-CTG	C	T	T	C	C	C	C*	C*	Chimpanzee		*TCG
21	GRIN3A	A119T	GCG-ACG	G	A	G	G	G	G	G	G	Chimpanzee
22	GRIN3A	A121T	GCC-ACC	G	A	A	A	A	A	A	A	Human	Fixed
23	GRIN3A	V138M	GTG-ATG	G	A	G	G	G*	G*	G*	G	Chimpanzee		*GGG
24	GRIN3A	E340K	GAA-AAA	G	A	G	G	G	G	G	G	Chimpanzee
25	GRIN3A	A885S	GCC-TCC	G	T	T	T	T	T	T	T	Human	Fixed
26	GRIN3A	I988V	ATA-GTA	A	G	G	G	G	G	G	G	Human	Fixed
27	GRIN3A	R1059L	CGG-CTG	G	T	T	-	T	T	T	T	Human	Fixed
28	GRIN3B	P17S	CCG-TCG	C	T	T	C	C	C	C	C	Chimpanzee
29	GRIN3B	G175S	GGC-AGC	G	A	G	-	G	G	G	G	Chimpanzee
30	GRIN3B	E229G	GAA-GGA	A	G	A	-	A	A	-	A	Chimpanzee
31	GRIN3B	A272V	GCG-GTG	C	T	C	C	C*	C	C*	C*	Chimpanzee		*GCA
32	GRIN3B	I296T	ATT-ACT	T	C	C	C*	C	C*	C/T**	C*	N.A.		*ACG,**ATT/CCT
33	GRIN3B	W414R	TGG-CGG	T	C	C	C	C	C	C	C	Human	NOT	rs2240157
34	GRIN3B	A468V	GCG-GTG	C	T	T	T	T	T	T	T	Human	Fixed
35	GRIN3B	R473C	CGC-TGC	C	T	T	C	C	C	C	C	Chimpanzee
36	GRIN3B	L499I	CTC-ATC	C	A	A	A	A	A	A	A	Human	Fixed
37	GRIN3B	T577M	ACG-ATG	C	T	T	T	T	-	T	T	Human	NOT	rs2240158
38	GRIN3B	Y595C	TAC-TGC	A	G	G	G	A	-	A	A	N.A.
39	GRIN3B	R598C	CGT-TGC	CGT	TGC	TGC	TGC	CGC	-	CGC	CGC	N.A.
40	GRIN3B	V613I	GTC-ATC	G	A	G	G	G	-	G	G	Chimpanzee
41	GRIN3B	R727H	CGC-CAC	G	A	A	A	A	A	"A"	"A"	Human	Fixed
42	GRIA3	P590L	CCT-CTT	C	T	C	C	C	C	C	C	Chimpanzee
43	GRIA4	S5C	TCC-TGC	C	G	G	G	G	G	G	G	Human	Fixed
44	GRIK3	S310A	TCC-GCC	T	G	G	G	"G"	G	G	G	Human	NOT	rs6691840
45	GRIK3	V419I	GTT-ATT	G	A	A	G	G	G	G	G	Chimpanzee
46	GRIK4	H403R	CAC-CGC	A	G	G	G	A	G	G	G	N.A.
47	GRIK5	L298P	CTG-CCG	T	C	C	C	C	C	C	C	Human	Fixed
48	GRIK5	I809V	ATC-GTC	A	G	G	G	"G"	-	G	G	Human	Fixed	*GTT
49	GRIK5	A922T	GCC-ACC	G	A	-	-	G	G	G	G	Chimpanzee
50	GRIK5	V956A	GTC-GCC	T	C	C/T	-	C/T	-	C	C	N.A.
51	GRID1	T295M	ACG-ATG	C	T	T	T	T	-	T	T	Human	Fixed
52	GRID1	M628V	ATG-GTG	A	G	G	G	G	G	G	G	Human	Fixed
53	GRID2	S11F	TCC-TTC	C	T	T	C	C	C	C	C	Chimpanzee
54	GRM1	S993P	TCC-CCC	T	C	C	C	C	C	C	C	Human	NOT	rs6923492
55	GRM1	L1089P	CTG-CCG	T	C	C	C	C	C	T	T	N.A.		*CCA
56	GRM2	A6G	GCG-GGG	G	C	C	C	C	C	C	C	Human	Fixed
57	GRM2	A248V	GCG-GTG	C	T	T	C	C	C	C	C	Chimpanzee
58	GRM3	M547V	ATG-GTG	A	G	G	A	A	A	A	A	Chimpanzee
59	GRM3	S551P	TCT-CCT	T	C	C	C	C	C	C	C	Human	NOT	No rs# available
60	GRM3	M593T	ATG-ACG	T	C/T	T	T	T	T	T	T	Chimpanzee
61	GRM4	L19F	CTC-TTC	C	T	T	C	C	C	C	C	Chimpanzee
62	GRM6	Q59P	CAG-CCG	A	C	C	C	C	C	C	C	Human	NOT	rs2645329
63	GRM6	P141T	CCC-ACC	C	A*	A	A	A	A	A	-	Human	NOT	No rs # available*AT/CC
64	GRM6	D380E	GAT-GAG	T	G	G	T	T	T	T	T	Chimpanzee
65	GRM6	M442T	ATG-ACG	T	C	C	C	C	T	C	C	N.A.
66	GRM6	Y612H	TAC-CAC	T	C	C	C	C	C	C	C	Human	Fixed
67	GRM6	A650G	GCG-GGG	C	G	G	C	C	C	T	T	N.A.
68	GRM6	M714V	ATG-GTG	A	G	G	G*	G	G	G	G	Human	Fixed	*GCG
69	GRM6	V839I	GTA-ATA	G	A	A	G	G	G	G	G	Chimpanzee
70	GRM6	A877D	GCC-GAC	C	A	A	A	A	A	A	A	Human	Fixed
71	GRM7	A520P	GCC-CCC	C	G	G	G	G	G	G	G	Human	Fixed
72	GRM8	R268C	CGC-TGC	A	G	A	A	A	A	A	A	Chimpanzee
73	GRM8	G327V	GGG-GTG	G	G/T	G/T	G	G	G	G	G	Chimpanzee
74	GRM8	V653I	GTC-ATC	G	A	A	A	A	A	A	A	Human	Fixed
75	GRIN2C	del1021-1026RALPER	CGCGCGCTCCCAGAGCGG	del	in	in	-	in	in	in	in	Human	Fixed
76	GRIN2C	PPE 1055-1057del	CCCCCGGAG	in	in/del	in	-	"in"	in	in	in	Chimpanzee
77	GRIN2C	AH1164-1165del	GCCCAC	in	in/del	in	in	in	in	in	-	Chimpanzee
78	GRM6	GD125-126del	GCGACG	in	del	del	-	del	del	del	-	Human	Fixed

Abbreviations are: Hum - human, Chimp - chimpanzee, Bon - bonobo, Gor - gorilla, Ora - orangutan, Gib - gibbon, in - insertion, del -- deletion, rs -- RefSNP accession ID.1: This shows the lineage in which the substitution (insertion/deletion) occurred. In cases in which the substitution independently occurred at the same site in more than two lineages, the specificity is not assigned (N.A.). We pooled substitutions found specifically in chimpanzees or in bonobos as "chimpanzee-specific" substitutions.

" ": The sequence was obtained from the UCSC genome database.

-: We failed to determine the sequence of the site, and the sequence was not available from the UCSC genome database.

*: The other substitution was found in the codon.

Functional implications of human-specific "fixed" nonsynonymous substitutions

To evaluate the functional significance of each amino acid substitution in the GluR genes, we calculated Grantham's distance [19], a measurement of the chemical drasticity of amino acid replacements. We examined the differences in amino acid substitution patterns between humans and chimpanzees using 35 human-specific and 29 chimpanzee-specific substitutions after excluding 4 indel sites. We classified amino acid substitutions into two groups: substitutions with Grantham's distances greater than 100 (the mean chemical distance from the three-property formula [19]) were classified as "radical" changes and substitutions with Grantham's distance less than 100 were classified as "non-radical" changes. We found four radical and 30 non-radical changes in the human lineage and four radical and 25 non-radical changes in the chimpanzee lineage, indicating that there are no significant differences in the amino acid substitution patterns between humans and chimpanzees (p = 1 in Fisher's exact test, 2 × 2, two-tailed). We then summarized Grantham's distance for each GluR gene that contained one of the 28 human-specific "fixed" substitutions (Table 4). Among the 11 GluR genes that have human-specific "fixed" substitutions, Grantham's distance analysis showed that GRIN2C and GRIN3A are the most diverged in humans from the human-chimpanzee ancestor sequence (476 in GRIN2C and 438 in GRIN3A). Four out of the five "fixed" substitutions in GRIN2C and five out of the six "fixed" substitutions in GRIN3A are non-radical changes with Grantham's distances of less than 100. These substitutions are not located in any particular region, but instead are distributed throughout the entire gene regions (Table 4). These results imply that it is unlikely that the accumulation of non-radical substitutions in GRIN2C and GRIN3A caused their functional divergence from their ancestors.

Table 4

Grantham's distance for GluR genes using human-specific "fixed" amino acid substitutions

Gene	Number of substitutions	Total Grantham's distance	Substitutions(Amino acid position, Grantham's distance)
GRIN2C	5	476	(89, 29) (100, 94) (596, 99) (933, 74) (1221, 180)
GRIN3A	6	438	(30, 56) (71, 94) (121, 58) (885, 99) (988, 29) (1059, 102)
GRM6	3	230	(612, 83) (714, 21) (877, 126)
GRIK5	2	127	(298, 98) (809, 29)
GRIN2A	3	125	(906, 46) (1006, 64) (1221, 15)
GRIA4	1	112	(5, 112)
GRID1	2	102	(295, 81) (628, 21)
GRIN3B	3	98	(468, 64) (499, 5) (727, 29)
GRM2	1	60	(6, 60)
GRM8	1	29	(653, 29)
GRM7	1	27	(520, 27)
Total	28	1824

Using the MEMSAT3 [20] and MyHits [21], we annotated the transmembrane and protein motif domains around the 30 "fixed" human-specific substitution/indel sites (Table 5). We found that ten substitutions are located within either transmembrane or protein motif domains: four in transmembrane domain sites, two in N-glycosylation sites, two in N-myristoylation sites, and two in phosphorylation sites. Out of these ten substitutions, there are two human-specific and "fixed" substitutions, D71H in GRIN3A and R27H in GRIN3B, which alter the functional assignments of their respective genes as determined by the aforementioned annotation software. D71G in GRIN3A abolishes an N-myristoylation site that is conserved in other apes and R727H in GRIN3B generates a novel phosphorylation site for protein kinase C in the human lineage. These substitutions may cause functional changes in human GluRs that contribute to human brain function.

Table 5

Human-chimpanzee amino acid substitutions in functional domains

Gene	Amino acid(Human-Chimpanzee)	Nucleotide(Human-Chimpanzee)	Functional Domain
GRIN2A	S906N	AGC-AAC	N-glycosylation site
GRIN3A	S30G	AGC-GGC	N-myristoylation site
GRIN3A	D71G	GAC-GGC	N-myristoylation site (lost in humans)
GRIN3A	I988V	ATA-GTA	Casein kinase II phosphorylation site.
GRIN3B	A468V	GCG-GTG	N-glycosylation site
GRIN3B	R727H	CGC-CAC	Protein kinase C phosphorylation site (acquired in humans)
GRIK5	I809V	ATC-GTC	Transmembrane
GRM2	A6G	GCG-GGG	N-myristoylation site
GRM6	Y612H	TAC-CAC	Transmembrane
GRM6	M714V	ATG-GTG	Transmembrane
GRM8	V653I	GTC-ATC	Transmembrane

Discussion

In this study, we examined the evolutionary changes of the glutamate receptor (GluR) genes in humans and chimpanzees. We found no gross differences in the coding regions or splice sites of the GluR genes between humans and chimpanzees. We also demonstrated that the average rate of protein evolution (i.e. the K/Kratio) is significantly lower in the GluR genes than the genome-wide average values for humans and chimpanzees. This pattern is consistent with previous genome-wide studies [15,22,23], indicating that strong purifying selection acts on brain-expressed genes including the GluR genes due to their strict functional constraint. There are no significant differences between humans and chimpanzees with regard to their K/Kvalues (Table 2) or substitution patterns (Table 4). These results imply that no gross functional changes occurred in either lineage after the human-chimpanzee divergence.

Dorus et al. [24] found an increase in the K/Kratio of genes involved in the nervous system of primates relative to rodents when housekeeping genes are treated as a control, leading to the conclusion that the primate nervous system genes have experienced accelerated evolution. Our results indicate similar K/Kratios between these primate nervous system genes and the GluR genes. Although our K/Kratio values for the GluR genes are higher than those of housekeeping genes as discussed in Dorus et al. [24], we conclude that the GluR genes have been subject to strong functional constraint rather than rapid positive selection detected as accelerated evolution for the following reasons: First, we could not detect any positive selection for the GluR genes using the statistical tests reported by Dorus et al. [24]. Although the statistical power of the tests may have been somewhat affected by the scarcity of substitutions, the improved branch-site test can detect single amino acid substitutions positively selected [18]. Second, there is no local accumulation of amino acid substitutions that could have caused the functional divergence of domains in the GluR genes. Grantham's distance analyses showed that GRIN2C and GRIN3A are the most and the second most diverged GluR genes between humans and chimpanzees. However, most of the substitutions in these genes are non-radical and are not clustered in any particular region. Third, we identified only two out of the 28 human-specific "fixed" substitutions in the assigned functional transmembrane region. This observation strongly supports severe functional constraint acting on the coding regions of the GluR genes, especially on the functionally important domains.

Niemann et al. [25] reported a common null allele of GRIN3B with no particular phenotype, indicating relaxed functional constraints on GRIN3B in the human lineage. However, we observed a low K/Kratio (0.2976) and a low Grantham's distance (98) for human GRIN3B. Gene loss or decay might still contribute to functional changes by modifying the genetic network. In fact, NR3B knockout mice have been reported to show highly increased social interaction with their cage mates in their home cage but moderately increased anxiety-like behavior and decreased social interaction in a novel environment [26]. The presence of NR3 in NMDA receptors has been shown to decrease Mg+2 sensitivity and Ca+2 permeability, reduce agonist-induced current responses, and give rise to a new class of excitatory glycine receptors [27]. These observations suggest that NR3 has a significant role in higher brain functions through tetrameric formation with other NR subunits. Two out of the 28 human-specific "fixed" substitutions, D71G in GRIN3A and R727H in GRIN3B, changed the functional assignments between humans and other apes, causing the loss of a myristoylation site and the gain of a phosphorylation site, respectively. Since myristoylation and phosphorylation are commonly involved in processes related to synaptic plasticity including long-term potentiation and long-term depression in glutamate receptors [28], these two substitutions possibly affect human-specific brain function by modulating NMDA receptor characteristics.

Conclusion

The results of our comparative genetic study enable us to speculate about the evolutionary changes affecting human-specific brain function that occurred in the GluR genes. We showed that strong purifying selection is the major evolutionary force in the GluR genes shared by humans and chimpanzees. We identified 30 human-specific "fixed" amino acid substitutions/indels including two amino acid substitutions that potentially alter the functional roles of their genes as candidate sites responsible for human-specific brain function. Our results are valuable for understanding the molecular basis of the brain and nervous system in humans and help us to clarify human GluR functions in in vitro and in vivo experiments.

Methods

Sequence data for human, chimpanzee and macaque

Using the longest isoform transcript as a reference (Additional file 3), we retrieved the coding sequences of 26 glutamate receptor (GluR) human and chimpanzee genes from the UCSC Genome Browser (hg18 and panTro2 for humans and chimpanzees, respectively)[12]. Since only partial genomic sequences were available for 21 chimpanzee genes, we determined the chimpanzee sequence for the 21 genes using either PCR-based direct sequencing or a BAC-based cloning-sequencing method (Additional file 3). These genomic GluR sequences were deposited in GenBank (accession numbers: AB514205-AB514225). The genomic sequences of macaque homologs were also obtained from the UCSC Genome Browser (rheMac2).

DNA samples

Primate DNA samples were kindly provided by Dr. Osamu Takenaka of the Primate Research Institute at Kyoto University and Dr. Takafumi Ishida from the Department of Biological Sciences at the Graduate School of Science of The University of Tokyo. Japanese samples and Thai samples were collected from the Kyushu area of Japan and the Chiang Mai area of Thailand with written informed consent. Other ethnic human samples were purchased from The Coriell Institute for Medical Research [29]. We identified human-specific substitution sites by determining the sequences of five primate species, the bonobo (Pan paniscus) gorilla (Gorilla gorilla ssp), orangutan (Pongo pygmaeus pygmaeus), Siamang (Symphalangus syndactylus), crab-eating macaque (Macaca fascicularis), and green monkey (Chlorocebus aethiops) at the human-chimpanzee substitution sites. Then, we analyzed 80 human DNA samples (Additional file 4), including 7 populations, to confirm "fixation" of these mutations in human species. This study was approved by the Ethics Committee of the Faculty of Medicine at Kyushu University.

PCR amplification and sequencing

The PCR primers were designed based on the alignments of human and chimpanzee GluR genes using Primer3 [30]. PCR amplification was carried out using 10 μl samples containing 1 μg of genomic DNA, PCR buffer, 2.5 mM dNTPs (Promega, Madison, WI), 0.7 units Taq DNA polymerase (Promega, Madison, WI), 25 mM MgCl2, and 10 μM forward and reverse primers. Primer pairs and PCR conditions are described in Additional file 5. After the PCR reaction, we treated the reaction mixtures with 1 U of Exonuclease I (New England Biolabs) and 0.1 U of SAP (Shrimp Alkaline Phosphatase; Roche Applied Science, Indianapolis, IN) to remove the primers.

All sequencing reactions were performed using 10 μl samples containing 1 μl of PCR product, 1.6 μM sequencing primer, and 0.25 μl of BigDye Terminator v1.1 or v.3.1 (Applied Biosystems, Foster City, CA). The conditions for the sequencing reaction were 96°C for 90 seconds, 50°C for 5 seconds, and 60°C for 60 seconds for 25 cycles. The sequencing products were purified by ethanol precipitation and then analyzed on an ABI 3100 or 3730 (Applied Biosystems). Mutation Surveyor v2.2 (SoftGenetics, LLC) was used to compile the electropherograms.

Data analysis

The pairwise nucleotide divergence was estimated by MEGA [31]. The Tamura and Nei model [32] and Jukes and Cantor model [33] were used for total and synonymous sites, respectively. Standard errors were computed by the bootstrap method (1000 replicates). The nonsynonymous and synonymous lineage-specific rates (Kand K, respectively) were estimated by the modified Nei and Gojobori [34] and ML [35] methods as implemented in the codeml module of PAML [16].

The Grantham's distances for each substitution were obtained from Table in [19]. Since this distance method is only applicable to amino acid substitutions, we excluded insertion/deletion (indel) sites from this analysis. We used the mean chemical distance from the three-property formula [19], 100, for the cutoff value of two classes, radical and non-radical substitution.

Using PAML software, the likelihood ratio test was applied to examine the following two hypotheses: (1) the K/Kratio was significantly higher in the branch of interest than in the background branch (tests B and D in [17]) and (2) positive selection acted on the branch of interest (improved branch site model; test 2 in [18]). The tests were carried out by comparing the log-likelihood values between the null and alternate hypotheses. Bonferroni correction was applied to correct for multiple comparisons.

The topology of transmembrane domains was predicted by the MEMSAT3 module [20] of The PSIPRED Protein Structure Prediction Server [36]. MyHits [21] with the PROSITE database [37] was used to scan all known protein motifs.

Authors' contributions

HS, HG, and YF designed the study. YF and HS collected human and primate DNA samples. YK, AT, MH, YS, and AF carried out the sequencing of the chimpanzee GluR genes. KW, NA, RK, KT, HS, and HG sequenced the human-chimpanzee substitution sites in humans and the other primates. HG and HS performed data analyses. HG, HS, and YF drafted the manuscript. All authors read and approved the final manuscript.

Additional file 1

The likelihood statistics used to compare the . The table shows likelihood ratios and p value for the statistical test of the K/Kratio comparison between human and background lineage.

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Additional file 2

Results for the improved branch site model. The table presents likelihood ratios and p value for the improved branch site test.

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Additional file 3

The source of genomic GluR sequences for humans, chimpanzees, and macaques. The table shows the data source of genomic GluR sequences.

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Additional file 4

Human DNA samples used in the polymorphism survey. The table presents Coriell numbers of human DNA samples for genotyping in human populations.

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Additional file 5

Primers and PCR conditions. The table shows the primer sequences and PCR conditions for genotyping in primates.

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