Table 2.
Substitutions of proline-258 in TMS VI of the α-factor receptor: effects on receptor signal transduction, agonist binding affinity, and cell surface expression
| STE2 allele |
FUS1-lacZ expression (% wild type
+ α-factor)
|
α-Factor binding sites | ||||
|---|---|---|---|---|---|---|
|
SST2
|
sst2Δ
|
SST2
|
||||
| −α-factor | +α-factor | −α-factor | +α-factor | Kd (nM) | Bmax (sites/cell) | |
| ste2Δ | 0.2 | 0.2 | 1.6 | 1.4 | − | − |
| wild type | 0.2 | 100 | 1.3 | 100 | 3.3 | 15,000 |
| P258A | 2.7 | 78 | 14 | 100 | 0.37 | 1,000 |
| P258I | 4.2 | 110 | 18 | 120 | 0.44 | 1,100 |
| P258L | 5.3 | 140 | 13 | 99 | 0.42 | 1,700 |
| P258M | 14 | 120 | 53 | 110 | 1.0 | 480 |
| P258V | 2.8 | 110 | 21 | 70 | 0.34 | 1,500 |
| P258D | 1.0 | 23 | 7.1 | 58 | 0.58 | 260 |
| P258E | 0.2 | 18 | 6.0 | 51 | 0.56 | 70 |
| P258H | 0.2 | 0.5 | 6.1 | 6.9 | n.d. | n.d. |
| P258K | 0.2 | 7.2 | 5.3 | 28 | 1.2 | 40 |
| P258R | 0.4 | 16 | 3.6 | 17 | 0.68 | 60 |
| P258C | 0.5 | 23 | 13 | 56 | 0.45 | 210 |
| P258G | 0.4 | 20 | 5.6 | 110 | 1.0 | 170 |
| P258N | 1.7 | 3.2 | 11 | 12 | n.d. | n.d. |
| P258Q | 0.6 | 10 | 3.5 | 9.4 | 0.58 | 40 |
| P258S | 0.3 | 0.6 | 4.6 | 7.3 | n.d. | n.d. |
| P258T | 0.3 | 0.7 | 6.8 | 7.8 | n.d. | n.d. |
| P258Y | 13 | 47 | 25 | 63 | n.d. | n.d. |
| P258F | 3.7 | 83 | 27 | 91 | 2.5 | 170 |
| P258W | 1.6 | 3.2 | 5.3 | 6.9 | n.d. | n.d. |
| 300ter | 1.0 | 79 | ||||
| P258L,300ter | 45 | 92 | ||||
The indicated STE2 alleles were expressed from centromeric plasmids (pRS314 derivatives) in isogenic MATa ste2Δ mfα1Δ mfα2Δ strains that expressed (KBY16) or lacked (KBY17) the SST2 gene. Cells also contained the pheromone-inducible FUS1-lacZ gene on plasmid pSL307. Cells were treated as indicated with synthetic α-factor (1 μM, 2 h at 30°), and activation of the pheromone response pathway was quantified by performing β-galactosidase assays. Data are expressed as the percent of the activity detected in α-factor–treated cells that expressed the wild-type STE2 gene. Data shown for each STE2 allele are the average obtained from assays of at least four independent transformants, each of which was assayed in duplicate; standard errors were 10-30% of the values shown. Radioligand binding assays were performed using [35S]α-factor and KBY16 cells expressing the indicated STE2 alleles from centromeric plasmids (pRS314 derivatives). The Kd and Bmax values shown for cells expressing each STE2 allele were calculated by nonlinear regression of data obtained from two to three independent transformants assayed in duplicate; standard errors for these determinations were 5-15% of the values shown. n.d., Specific binding was not detected.