Purification and characterization of the human brain insulin receptor.

R A Roth,V Sara,D O Morgan,J Beaudoin

doi:10.1016/s0021-9258(17)35711-3

Abstract

The insulin receptor from human brain cortex was purified by a combination monoclonal antibody affinity column and a wheat germ agglutinin column. This purified receptor preparation exhibited major protein bands of apparent Mr = 135,000 and 95,000, molecular weights comparable to those for the alpha and beta subunits of the purified human placental and rat liver receptors. A minor protein band of apparent Mr = 120,000 was also observed in the brain receptor preparation. Crosslinking of 125I-insulin to all three receptor preparations was found to preferentially label a protein of apparent Mr = 135,000. In contrast, cross-linking of 125I-labeled insulin-like growth factor I to the brain preparation preferentially labeled the protein of apparent Mr = 120,000. The purified brain insulin receptor was found to be identical with the placental insulin receptor in the amount of neuraminidase-sensitive sialic acid and reaction with three monoclonal antibodies to the beta subunit of the placental receptor. In contrast, a monoclonal antibody to the insulin binding site recognized the placental receptor approximately 300 times better than the brain receptor. These results indicate that the brain insulin receptor differs from the receptor in other tissues and suggests that this difference is not simply due to the amount of sialic acid on the receptor.

Highlights

M, = 135,000.In contrastc, ross-linkingof 12%labeled and cross-linking of ‘251-insulinand autophosphorylation to insulin-like growth factor I to the brain preparation examine the structure of the a and /3 subunits, respectively. preferentially labeled the protein of apparent M, = Via these techniques,the structureof the insulin receptor has afi1non2ud0tnh,d0ere0aat0moc,toiTubonhnewetpiidutohfernitnfhtiieewrcdueabietlrrhaamtmihnoieinnnosipuc~llloai~nncreeae-nlcsteeaapnnltstoiiirbntoisvsduieilaeinlsictroaweccatie,hfd?spetoeabrrneyedtnhberrxoaacimyntien(s3e1(d2-377i,n)2.f8a)Ot,fm(to9hne, os2en2u-v2ca4lre)i,aorumcseutlsilcsslse(u2e9(s2),5ol)i,nvlkeyridt(nh7e,ey8b,(rs2ao6i)n), subunit of the placental receptorI.n contrast, amono- insulin receptor has been reported to differ fromthe placental clonal antibody to the insulin binding site recognized receptor
Mostinterestingwas the finding that themonoclonal antibody tothe insulinbinding site inhibited'2sII-insulin binding to thepurified brain receptor preparation with about 300 times less potency than itinhibited insulin binding to the purified placental receptor (Fig. 1B)
The ability of two other monoclonal antibodies to interact with the brainreceptor was examined by a radioimmunoassay for the insulin receptor (54).These two other antibodies have been shown to bind to distinct antigenic regions of the placental /3 subunit (47)

Summary

Purification and Characterization of thHe uman Brain Insulin Receptor*

From the $Department of Pharmacology, Stanford University School of Medicine, Stunford, California 94305 the fDepartment of Psychiatry, Kurolinska Intitute, Stockholm, Sweden. M , = 135,000.In contrastc, ross-linkingof 12%labeled and cross-linking of ‘251-insulinand autophosphorylation to insulin-like growth factor I to the brain preparation examine the structure of the a and /3 subunits, respectively. The/3 subunit ispredominantly labeled whenthe receptor is labeledby either an intrinsic kinase activity of the receptor (10-13) or when ATP analogs are covalentlycoupled to thereceptor (14-16) Both subunits areglycosygel electrophoresisand comparedto theplacental receptor for its ability to interact with a group of monoclonal antibodies to the placental insulin receptor (46-48). The complete amino acid sequence of digestion and the structure of the

EXPERIMENTAL PROCEDURES

RESULTS

CONCENTRATION fMI

DISCUSSION