Distinct biochemical properties of the native members of the G12 G-protein subfamily. Characterization of Gα12 purified from rat brain

Abstract

G12 and G13 are insufficiently characterized pertussis toxin-insensitive G-proteins. Here, we describe the isolation of G alpha 12 from rat brain membranes. G alpha 12 was purified to apparent homogeneity by three steps of conventional chromatography, followed by two cycles of subunit-exchange chromatography on immobilized G subunits. Purified G alpha 12 bound guanosine 5'-[gamma-thio]triphosphate slowly and substoichiometrically. For isolation of functionally active G alpha 12, it was mandatory to use sucrose monolaurate as a detergent. Comparative studies of both rat-brain-derived members of the G12 subfamily revealed differences in the affinity of G alpha 12 and G alpha 13 for G beta gamma. G alpha 12 required a higher Mg2+ concentration for AlF4- -induced dissociation from immobilized G beta gamma than did G alpha 13. In addition, the G12 subfamily members differed in their sedimentation velocities, as determined by sucrose-density-gradient centrifugation. Analysis of sedimentation coefficients revealed a higher tendency of G12 to form supramolecular structures in comparison to G13 and other G-proteins. These G13 structures were stabilized by sucrose monolaurate, which in turn may explain the necessity for this detergent for purification of functionally active G alpha 12. Despite these distinct biochemical characteristics of G12 and G13, both purified G-proteins coupled to a recombinant thromboxane A2 (TXA2) receptor reconstituted into phospholipid vesicles. These data indicate, (1) significant differences in the biochemical properties of native members of the G12 subfamily, and (2) their specific coupling to TXA2 receptors.

Investigation of the role of the carboxyl-terminal tails of the α and β isoforms of the human thromboxane A2 receptor (TP) in mediating receptor:effector coupling

“…A number of independent studies have implicated TP coupling to members of the G 12 family [21][22][23][24]54] and the presence of both Gα 12 and Gα 13 in platelets and other hematopoietic cells has been widely…”

The G-protein G13 but Not G12 Mediates Signaling from Lysophosphatidic Acid Receptor via Epidermal Growth Factor Receptor to Rho

“…In addition, these antisera crossreacted with several unknown proteins of higher and lower molecular masses that had not been detected in rodent and nonrodent brain membranes (17). To assure the specificity of AS 232 and AS 233, which have been extensively characterized (17,18), we performed peptide competition experiments. Upon preincubation with the peptide used for their generation, the detection of G␣ 12 was abolished (data not shown).…”

“…The subtype specific antisera AS 232 and AS 233 (anti-␣ 12 ); AS 343, AS 342, and AS 272 (anti-␣ 13 ); AS 369 (anti-␣ q/11 ), and AS 266 (anti-␣ i ) were characterized previously (17). Antisera against G␣ 12 were incubated with the peptides (10 g/ml) used for their generation, as published elsewhere (17,18). Detection of filter-bound proteins was carried out using the enhanced chemiluminescence (ECL) Western blotting system purchased from Amersham (19).…”

“…32 P]GTP azidoanilide (3 Ci/tube; specific activity 3,000 Ci/mmol) at 30°C for different times: 3 min for G␣ i , 10 min for G␣ q/11 , and 30 min for G␣ 12 and G␣ 13 (18). Samples were irradiated for 15 s at 4°C with a 254-nm UV lamp.…”