TABLE 1

Summary of binding, Gαi-, and Gβγ-coupling properties of 5-HT1A-i2 mutants

Agonist-induced changes in intracellular free calcium concentration in Ltk- cells transiently transfected with wild-type 5-HT1A (1A) or 5-HT1A-i2 mutant receptors were measured and quantified as the percentage of control over basal [Ca2+]i levels (column 1). In parallel experiments, membranes were prepared from Ltk- cells transiently transfected with the indicated 5-HT1A mutant receptors and were subjected to binding analysis with [3H]8-OH-DPAT (column 2). Agonist-independent Gβγ-mediated activation of adenylyl cyclase II was determined using HEK 293 cells transiently transfected with expression plasmids for adenylyl cyclase II, Gαi2, and the indicated wild-type (1A) or i2 mutant 5-HT1A receptor. Agonist-independent cAMP production was determined using the average difference between pertussis toxin-treated and nontreated cells for three to six independent experiments, where (+++) indicates ≥80%, (++) ≥50%, and (+) between 19 and 27% increase in cAMP production compared with wild-type 5-HT1A receptor, and - indicates no detectable activity (column 3). For Gαi-mediated inhibition of adenylyl cyclase, HEK cells were transiently cotransfected with 12 μg of D1 receptor cDNA and an equal amount of wild-type (1A) or 5-HT1A-i2 mutant receptors. Inhibition of D1/Gαs-stimulated (10 μM apomorphine) cAMP accumulation by 5-HT1A agonist 8-OH-DPAT (1 μM) was measured by specific radioimmunoassay, and data were presented as the percentage of inhibition of D1-stimulated cAMP levels (column 4). In all cases, data are expressed as mean ± S.E.M. of at least three independent experiments, and values that are significantly different from 0 were identified using Student's paired t test and are indicated as ***, p < 0.001; **, p < 0.01; *, p < 0.05.

Substitution DPAT-Induced [Ca2+]i (percentage over basal [Ca2+]i) Receptor Number Constitutive Activity (Level of Coupling to βγ-Mediated Stimulation of ACII) Inhibition of D1-Stimulated cAMP Accumulation
% pmol/transfection %
WT (1A) 101 ± 8** 1.9 ± 0.4* +++ 48 ± 9*
T149A (i2) 27 ± 9* 2.3 ± 0.7* + 53 ± 10*
D143F 15 ± 3* 1.5 ± 0.9 − 54 ± 8**
D143V 22 ± 4* 2.5 ± 0.2** − 56 ± 9**
D143N 26 ± 7* 0.9 ± 0.1 ** − 45 ± 9*
D143L 29 ± 7* 0.7 ± 0.4 − 52 ± 3**
D143C N.R. 2.9 ± 0.8* − 44 ± 12*
Y144H N.R. 1.9 ± 0.2** − 22 ± 3**
Y144A 4 ± 2 1.7 ± 0.5* +++ 11 ± 8
Y144N 25 ± 7* 2.4 ± 0.6* − 44 ± 5**
Y144F N.R. 2.3 ± 0.6* ++ 43 ± 7**
Y144V 10 ± 6 2.3 ± 0.2** − 7 ± 2*
Y144I N.R. 1.4 ± 0.7 − 26 ± 5*
Y144C N.R. 2.1 ± 1.1 − −20 ± 3**
V145L 26 ± 3** 2.2 ± 0.9 + 34 ± 11*
V145K 23 ± 9* 1.4 ± 0.2** + 41 ± 13*
V145Y 4 ± 3 1.3 ± 0.7 − −30 ± 10*
V145R N.R. 1.6 ± 0.6* − 15 ± 9
V145Q N.R. 1.9 ± 0.2** − 27 ± 7*
V145E 12 ± 5 1.8 ± 0.5* − 32 ± 9*
V145S N.R. 1.5 ± 0.6* − −12 ± 7
V145W N.R. 1.2 ± 0.2** − 17 ± 6*
N146T 56 ± 17* 1.8 ± 0.3** ++ 31 ± 8*
N146F 14 ± 5* 1.9 ± 0.7* − 42 ± 10*
N146A 20 ± 13 1.0 ± 0.2* − 53 ± 10*
K147R N.R. 2.3 ± 0.6* − −3 ± 6
K147Q N.R. 2.5 ± 1.2 − −11 ± 9
R148L 17 ± 5* 2.5 ± 0.4** − 13 ± 4*
R148Q 16 ± 2** 1.3 ± 0.2** − 20 ± 8*
R148K 12 ± 6 2.1 ± 0.1*** − 4 ± 10
R148E 28 ± 4** 1.5 ± 0.1** − 20 ± 3**
R148P N.R. 3.2 ± 0.9* + 21 ± 4**
R148G 19 ± 6* 2.8 ± 1.1* + 53 ± 10*
R148V 20 ± 9 1.2 ± 0.4* − 45 ± 8*
T149E 20 ± 4* 1.0 ± 0.4* + 50 ± 3**
T149V 10 ± 5 1.7 ± 1.0 − 46 ± 14*
T149G 80 ± 8** 3.3 ± 0.6* ++ 52 ± 8**
T149R 50 ± 3** 1.5 ± 0.9 +++ 60 ± 8**
T149Q 22 ± 2** 1.8 ± 0.8 + 39 ± 4**
T149M 15 ± 7 1.9 ± 0.2** − 42 ± 4**
T149W 9 ± 7 2.0 ± 0.7* − 44 ± 5**
T149P 6 ± 9 2.6 ± 0.4** ++ 27 ± 9*
P150R 2 ± 5 2.3 ± 0.1*** − −6 ± 3
P150N N.R. 1.3 ± 0.8 − −12 ± 1**
P150D N.R. 1.3 ± 0.2** − 4 ± 2
P150G N.R. 2.0 ± 1.0 − −6 ± 1**
P150I 7 ± 8 1.7 ± 0.2** − 4 ± 1*
P150L N.R. 0.9 ± 0.6 − −23 ± 9*
P150F N.R. 1.9 ± 0.5* − 15 ± 11
P150S N.R. 0.8 ± 0.1** − −19 ± 10
P150T N.R. 2.1 ± 0.8* − −48 ± 9*
R151Q 4 ± 6 1.7 ± 0.8 − 22 ± 8*
R151A 26 ± 4** 2.3 ± 0.9* + 24 ± 8*
R151M 8 ± 1** 2.1 ± 1.0 − 20 ± 4*
R151T 27 ± 3** 1.0 ± 0.1** + 38 ± 7*
R151L 2 ± 5 0.8 ± 0.2* − 30 ± 6*
R151K 2 ± 4 1.5 ± 1.1 − 26 ± 6*
R152N 16 ± 4* 1.4 ± 0.6 − 10 ± 7
R152V 8 ± 7 1.3 ± 0.5* − 37 ± 8*
R152A 13 ± 7 2.2 ± 0.9* − 56 ± 7**
R152P 26 ± 3** 1.1 ± 0.5 − 12 ± 11
R152D 27 ± 6* 1.5 ± 0.8 − −11 ± 2

  • N.R., no response to treatments.