Abstract
A somatostatin (SRIF) receptor and its associated Gi regulatory proteins was purified from GH4C1 rat pituitary cells by: 1) saturation of the membrane-bound receptor with biotinyl-NH-[Leu8,D-Trp22,Tyr25] SRIF28 (bio-S28); 2) solubilization of receptor-ligand (R.L) complex with deoxycholate-lysophosphatidylcholine (D.L); 3) adsorption of solubilized receptor-ligand complex to immobilized streptavidin; and 4) elution of receptor and G-protein by GTP. The receptor, a glycoprotein with an average M(r) of 85,000, was then purified to substantial homogeneity on immobilized wheat germ agglutinin. The 85-kDa glycoprotein was identified as a SRIF receptor by several criteria. (a) It had the same size as the chemically cross-linked R.[125I]L complex. (b) Yield of the purified protein increased and plateaued in the same range of bio-S28 concentrations where specific high affinity binding reached saturation. (c) It was copurified with appropriate G-protein subunits. The 85-kDa receptor and two other proteins with M(r) values of 35,000 and 40,000, the sizes of G beta and G alpha, did not appear in eluates from control streptavidin columns done with SRIF receptors loaded with nonbiotinylated S14. The 40-kDa protein was identified as a Gi alpha by ADP-ribosylation from [32P]NAD catalyzed by pertussis toxin. (d) Both the chemically cross-linked R.[125I]L complex and SRIF receptor purified from [35S]methionine-labeled GH4C1 cells were reduced in size to about 38 kDa by endoglycosidase F. (e) Amino acid sequence from the purified receptor was nearly identical with that of a recently cloned SRIF receptor subtype.
Highlights
SRIF28; 2) solubilization of receptor-ligand S14 is derived fromS28 by post-translational proteolytic (R-L) complex with deoxycholate-lysophosphatidyl- cleavage [6, 7]
The 85-kDa glycoprotein was identified as a SRIF receptor by several criteria. (a)It had the same size as the chemically cross-linked
One of the clearest examplesof tissue specificity is that two octapeptide SRIF analogshave over 1000-fold higher affinity for pituitary thanfor brain SRIF receptors[10]
Summary
Solubilization of a Complex betweenSRIFReceptor and ~‘2sI-Tyr’’]S14-Because the SRIF receptor was difficult to solubilize in active form from GH4C1membranes, we solubilized a receptor-radioligand complex after binding of radioligand to the membrane bound receptor. R, after chemical cross-linking as in A , membranes were solubilized in 0.15% D:L as in Table I and the 100,000 X g supernat,ants were incubated with WGA-agarose as described in the legend to Table I except that 4 mM TAC was added to one sample. In the WGA non-bound fraction, Inourinitialpurifications,the 35- and 40-kDa proteins a band with M, about 40,000 showed ligand specificity and a were the only ligand-specific species seen in the streptavidin clear correlation with the binding curve (Fig. C ) .There was eluates. N,N',N"-triacetylchitotriose (TAC).TheTACeluateconbio-S28 but not when bio-S28 binding was blocked with 10 tained nearly homogeneous 85-kDa protein (Fig. 58) At this P M S14 (Fig. 8A).The :'"S-labeled receptor was further puripoint the 85-kDa proteincould be stained by a normal silver fied on WGA-agarose and incubatedwith neuraminidase method, possibly due to reduced background and the ability t o load more material ona gel. Immunoblotting with anti-Gi, and anti-(;,, antisera has further confirmed that the 40- and 35-kDa proteins are Gi,, and G,,, respectively.:'
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