A lobster phospholipase C-beta that associates with G-proteins in response to odorants

Abstract

A cDNA clone encoding a protein of 1116 amino acids with significant homology to beta-isoforms of phospholipase C was isolated from lobster olfactory organ cDNA libraries and named lobPLCbeta. This cDNA hybridized predominantly to a 9 kb transcript in RNA from olfactory organ, pereiopod, brain, and eye-eyestalk and to several smaller minor transcripts only in eye-eyestalk. An antiserum raised to the C terminus of lobPLCbeta detected immunoreactivity in a single 130 kDa band in olfactory aesthetasc hairs, olfactory organ, pereiopod, dactyl, and brain. In eye-eyestalk this 130 kDa band was abundant, and minor bands of 100, 79, and 57 kDa also were detected. In cross sections of the aesthetasc hairs, immunoreactivity was detected in the outer dendritic segments of the olfactory receptor neurons, the site of olfactory transduction. A complex odorant caused lobPLCbeta immunoreactivity to increase in membrane fractions and decrease in soluble fractions of homogenates of aesthetasc hairs. The odorant also increased the amount of lobPLCbeta in immunoprecipitates of Galphaq and Gbeta from homogenates of aesthetasc hairs. These results support the conclusion that lobPLCbeta mediates olfactory transduction.

Fig. 1.

Comparison of the predicted amino acid sequence of lobPLCβ (GenBank accession number AF128539) with that of rat PLC-β4 and Drosophila NorpA (GenBank accession numbers 435757 and J03138). *Consensus PKC phosphorylation site;^¥consensus PKA phosphorylation site;^#consensus PKA and PKC phosphorylation site.lPLCβ, lobPLCβ; (… ), residues identical to lobPLCβ; (- - -), spaces introduced to improve alignment.

Fig. 2.

General features of the lobPLCβ modular structure. PH, Pleckstrin homology domain;EF, EF hand homology domain; X,Y, the bipartite catalytic domain; C2, C2 homology domain; P, P box; G, G-box required for functional interaction with G_αq.Underlined are the locations of polypeptide PLC293ct and of the conserved site of stimulatory interaction with G_βγ. aa, Amino acids.

Fig. 3.

Northern blot detection of lobPLCβ transcripts.Arrows mark bands at 9, 7, 5, 4, and 3 kb. Each lane contains 2 μg of poly (A)⁺ RNA isolated from the indicated lobster tissue. E, Eye-eyestalk; L, pereiopod; B, brain;N, olfactory organ. Size markers are given in kilobases.

Fig. 4.

Western blot of lobPLCβ, using antisera P293. Each lane contains 20 μg of membrane protein, except for eye-eyestalk, which contains 5 μg. The right panel shows a separate comparison of lobPLCβ immunoreactivity in lanes containing equal amounts of brain and aesthetasc hair membrane protein.A, Aesthetasc hair; B, brain;D, dactyl; E, eye-eyestalk;L, pereiopod; N, olfactory organ.E(s), Short exposure (2 sec) of the eye-eyestalk lane. Size markers are given in kilodaltons.

Fig. 5.

Immunoreactivity for lobPLCβ in the outer dendritic segments of olfactory receptor neurons in cross sections from the outer 500 μm of aesthetasc hairs. A, Staining with P293 antiserum. B, Staining with P293 antiserum preabsorbed with its antigen. C, Omission of P293 from the staining procedure. Scale bar, 20 μm.

Fig. 6.

The membrane association of lobPLCβ immunoreactivity increased after odorant stimulation. A, Western blot of membrane and supernatant fractions of an aesthetasc hair homogenate. B, Densitometry of replicate experiments shown in A (n = 2). Error bars indicate SEMs. C, Western blot for lobPLCβ in fractions of an eye-eyestalk homogenate. d, Darkness;l, light (room illumination); c, vehicle control; g, 3 μm GTP-γ-S;t, TetraMarin extract; sup, supernatant fraction.

Fig. 7.

Stimulation increased the association of lobPLCβ with G_αq and G_β. A, LobPLCβ immunoreactivity in G_β and G_αqimmunoprecipitates from aesthetasc hair homogenates. B, Densitometry of replicate experiments shown in A(n = 3 for all treatments, exceptn = 2 in lane t in G_βimmunoprecipitate). Error bars indicate SEMs. C, LobPLCβ immunoreactivity in G_β and G_αqimmunoprecipitates from eye-eyestalk homogenates. c, Vehicle control; t, TetraMarin extract;g, 3 μm GTP-γ-S; d, darkness; l, light (room illumination). *Significant difference from control by test of meaningfully paired comparisons;p < 0.05; df = 2).