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Certain peptides derived from the α1 domain of the major histocompatibility class I antigen complex (MHC-I) inhibit receptor internalization, increasing the steady-state number of active receptors on the cell surface and thereby enhancing the sensitivity to hormones and other agonists. These peptides self-assemble, and they also bind to MHC-I at the same site from which they are derived, suggesting that they could bind to receptor sites with significant sequence similarity. Receptors affected by MHC-I peptides do, indeed, have such sequence similarity, as illustrated here by insulin receptor (IR) and insulin-like growth factor-1 receptor. A synthetic peptide with sequence identical to a certain extracellular receptor domain binds to that receptor in a ligand-dependent manner and inhibits receptor internalization. Moreover, each such peptide is selective for its cognate receptor. An antibody to the IR peptide not only binds to IR and competes with the peptide but also inhibits insulin-dependent internalization of IR. These observations, and binding studies with deletion mutants of IR, indicate that the sequence QILKELEESSF encoded by exon 10 plays a key role in IR internalization. Our results illustrate a principle for identifying receptor-specific sites of importance for receptor internalization, and for enhancing sensitivity to hormones and other agonists.

We showed previously that peptides derived from the alpha 1 domain of the major histocompatibility complex class I protein (MHC-I) inhibit internalization of some receptors, thereby increasing the steady-state number of active receptors on the cell surface. In consequence, sensitivity to hormone (e.g., insulin) is enhanced, transport (e.g., of glucose by GLUT-4) is increased, and carrier proteins (e.g., transferrin) operate less efficiently. Now we report that a bioactive peptide (but not closely related inactive ones) binds to MHC-I on the cell surface, not in the groove but apparently to the alpha 1 helix. The binding is saturable, and the number of peptide binding sites on the cell surface approximately equals the number of MHC-I molecules. Antibodies to MHC-I inhibit peptide binding. Most significant, antibodies to MHC-I mimic the effect of a bioactive peptide, inhibiting receptor internalization. These results indicate that MHC-I participates in the regulation of cell surface receptor activity.

The stimulatory activity of peptides from the alpha 1 domain of the major histocompatibility complex (MHC) class I antigen on adipose cell glucose transport was previously shown to require a preformed, ordered conformation of the peptide. The two peptides studied previously were Dk-(61-85) (ERETQIAKGNEQSFRVDLRTLLRYY) and Dk-(69-85). We now show that systematic alanine substitution in Dk-(69-85) identifies residues that are essential for biological activity. Ordered structure of the peptides, estimated by circular dichroism, was found in all peptides with activity, but with a complex variety of spectra. Inactive peptides were in either a random coil or an ordered structure. Ordered structure, therefore, is not sufficient for activity. The peptides self-interact in the absence of cells and form aggregates that precipitate upon centrifugation. The tendency to aggregate is correlated with biological potency. Only MHC class I molecules have significant homology to the peptides studied here. The peptide self-interaction suggests that the biological effects in cells, which result from inhibition of receptor and transporter internalization, may be due to the binding (tantamount to self-interaction) of the peptide to the homologous sequences in the alpha 1 domain of the MHC class I molecule.

Peptides from the alpha 1 region (residues 61-85) of the D and K molecules of the major histocompatibility complex class I antigens inhibit insulin-induced tyrosine kinase activity of the purified human insulin receptors (IRs) as measured both by autophosphorylation and IR-mediated substrate [poly(Glu,Tyr)] phosphorylation. Half-maximal effect of the Dk-(61-85) peptide on IR autophosphorylation is obtained at 1.2 microM, and almost complete inhibition of IR kinase activity is obtained at 10 microM peptide. The corresponding K kappa-(61-85) peptide has a significantly weaker effect on autophosphorylation. No such effects are observed with nine peptides of similar length, but unrelated to major histocompatibility complex class I antigens. Neither of the major histocompatibility complex class I-derived peptides has any effect on the constitutively active kinase of a genetically engineered cytoplasmic IR domain. Further, insulin binding to IR is unaltered in the presence of the major histocompatibility complex class I-derived peptides. The inhibitory activity of the peptides on insulin-induced IR phosphorylation facilitated the observation that IRs require insulin to become substrate for an independent tyrosine kinase. In the presence of an inhibitory peptide, the constitutively active cytoplasmic IR kinase domain only phosphorylates the intact IR in the presence of insulin. We conclude that the tyrosine kinase activity of IRs may be altered by peptide interaction at an allosteric site and, moreover, IRs require insulin to assume a conformation permitting phosphorylation by an independent kinase.

We report the establishment of human-human hybridomas producing monoclonal antibody of predefined antigenic specificity. The U-266 human myeloma cell line was incubated in the presence of 8-azaguanine, and a rapidly growing, 8-azaguanine-resistant, hypoxanthine/amethopterin/thymidine (HAT) medium-sensitive mutant line, U-266AR1, was selected. These cells were fused with lymphoid cells from uninvolved spleens removed at staging laparotomy from patients with untreated Hodgkin's disease who had been previously sensitized to the chemical allergen 2,4-dinitrochlorobenzne. Hybrid cell cultures growing in HAT medium were screened for IgG production. Positive cultures were selected and their supernatants were tested in a solid-phase radioimmunoassay for reactivity with dinitrophenyl hapten coupled to bovine serum albumin. Cultures producing specific antibody were subcloned and expanded, and their antibody products were shown to be monoclonal by biosynthetic labeling and sodium dodecyl sulfate/polyacrylamide gel electrophoresis.