Pheromone binding by polymorphic mouse major urinary proteins

Abstract

Mouse major urinary proteins (MUPs) have been proposed to play a role in regulating the release and capture of pheromones. Here, we report affinity measurements of five recombinant urinary MUP isoforms (MUPs-I, II, VII, VIII, and IX) and one recombinant nasal isoform (MUP-IV) for each of three pheromonal ligands, (+/-)-2-sec-butyl-4,5-dihydrothiazole (SBT), 6-hydroxy-6-methyl-3-heptanone (HMH), and (+/-)dehydro-exo-brevicomin (DHB). Dissociation constants for all MUP-pheromone pairs were determined by isothermal titration calorimetry, and data for SBT were corroborated by measurements of intrinsic protein fluorescence. We also report the isolation of MUP-IV protein from mouse nasal extracts, in which MUP-IV mRNA has been observed previously. The affinity of each MUP isoform for SBT (K(d) approximately 0.04 to 0.9 micro M) is higher than that for DHB (K(d) approximately 26 to 58 micro M), which in turn is higher than that for HMH (K(d) approximately 50 to 200 micro M). Isoforms I, II, VIII, and IX show very similar affinities for each of the ligands. MUP-VII has approximately twofold higher affinity for SBT but approximately twofold lower affinity for the other pheromones, whereas MUP-IV has approximately 23-fold higher affinity for SBT and approximately fourfold lower affinity for the other pheromones. The variations in ligand affinities of the MUP isoforms are consistent with structural differences in the binding cavities of the isoforms. The data indicate that the concentrations of available pheromones in urine may be influenced by changes in the expression levels of urinary MUPs or the excretion levels of other MUP ligands. The variation in pheromone affinities of the urinary MUP isoforms provides only limited support for the proposal that MUP heterogeneity plays a role in regulating profiles of available pheromones. However, the binding data support the proposed role of nasal MUPs in sequestering pheromones and possibly transporting them to their receptors.

Fig. 1.

Pheromonal ligands used in this study: (±)-2-sec-butyl-4,5-dihydrothiazole (SBT), 6-hydroxy-6-methyl-3-heptanone (HMH), and (±)dehydro-exo-brevicomin (DHB).

Fig. 2.

Isothermal titration calorimetry data for binding of (±)-2-sec-butyl-4,5-dihydrothiazole (SBT) to MUP-I (left), binding of SBT to MUP-IV (center), and binding of 6-hydroxy-6-methyl-3-heptanone (HMH) to MUP-IV (right) at 30°C. Top panels are the raw data, and bottom panels are the fitted binding isotherms. Fitted K_d values were 0.90, 0.039, and 199 μM, respectively.

Fig. 3.

Three-dimensional graphical representation of the measured dissociation constants for each MUP-pheromone pair. For ease of visual comparison, K_d values for MUP–(±)-2-sec-butyl-4,5-dihydrothiazole (SBT) binding have been multiplied by a factor of 20.

Fig. 4.

Binding of (±)-2-sec-butyl-4,5-dihydrothiazole (SBT) to MUP-I (A) and MUP-IV (B), determined by intrinsic protein fluorescence. Each curve shows the ratio of fluorescence emission intensities at wavelengths of 303 and 337 nm plotted against the total SBT concentration. Protein concentrations were 1.0 μM (MUP-I) and 0.1 μM (MUP-IV), and the excitation wavelength was 228 nm. The fitted binding isotherms yielded K_d values of 2.5±1.9 and 0.32±0.29 μM, respectively. Note that the x-axis scale differs by a factor of 10 between the two panels.

Fig. 5.

Sequence alignment of the six MUP isoforms included in this study. Amino acids that differ from the corresponding residue in MUP-I are boxed. Positions of MUP-I residues with side-chains that show NOEs to (±)-2-sec-butyl-4,5-dihydrothiazole (SBT; Zidek et al. 1999) are indicated by arrows. The N-terminal sequence determined by Edman sequencing of nasal MUP-IV is underlined. Internal MUP-IV tryptic fragments observed by mass spectrometry are indicated in bold; the internal fragment sequenced by mass-spectrometric postsource decay analysis is also underlined.