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Role of the β93-100 Determinant Loop Sequence in Receptor Binding and Biological Activity of Human Luteinizing Hormone and Chorionic Gonadotropin

Endocrine Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School Boston, Massachusetts 02114
Department of Biochemistry and Molecular Biology, Mayo Medical School Rochester, Minnesota 55905

Address requests for reprints to: Henry T. Keutmann, M.D., Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114.

Abstract

The intercysteine loop sequence (93–100) in the β-subunit has been postulated to be important for receptor binding and specificity in the glycoprotein hormones, LH and human CG (hCG). To demonstrate this directly, and to characterize the structural features essential for activity, we prepared a series of synthetic peptides and analogs incorporating this determinant loop region. Peptides were assayed for inhibition of labeled hCG binding to ovarian membrane receptors and stimulation of testosterone production in Leydig cells. Peptides with the native (93–100) sequence from hCG and hLH inhibited hCG binding half maximally at 2.18 and 2.62 x 10^–4 M, respectively, while the sequence from FSH was inactive. Isosteric substitution of Ala for Cys resulted in an inactive peptide, indicating that the (93–100) disulfide bridge is essential for activity. Optimal binding activity requires at least one net positive charge among the side chains, as shown by loss of activity in hybrid analogs with neutral or negative charges conferred by progressive replacement of Arg by Asp at 94 and 95 or by introduction of Asp at 96 and 97. Despite binding to receptors, the native sequence did not promote testosterone production at doses up to 10^–2 M. This contrasts with a second receptor binding sequence, β(38–57) that activates testosterone production. There are differences between the (93–100) and (38–57) loop sequences in their chemical and physical properties, biological activity and antigenicity. While the cumulative evidence suggests that they associate with counterpart sites in -subunit to form a topographical binding domain in the whole hormone, our results suggest that each sequence may contribute in different ways to activation of postreceptor events.

FOOTNOTES

This work was supported by Grants HD-09140 and HD-12851 from the NIH.

Received for publication November 7, 1988. Revision received December 19, 1988. Accepted for publication December 19, 1988.

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