A conserved region in .alpha.-macroglobulins participates in binding to the mammalian .alpha.-macroglobulin receptor

Abstract

Efforts to characterize the receptor recognition domain of .alpha.-macroglobulins have primarily focused of human .alpha.2-macroglobulin (.alpha.2M). In the present work, the structure and function of the .alpha.-macroglobulin receptor recognition site were investigated by amino acid sequence analysis, plasma clearance, and cell binding studies using several nonhuman .alpha.-macroglobulins: bovine .alpha.2M, rat .alpha.1-macroglobulin (.alpha.1M), rat .alpha.1-inhibitor 3 (.alpha.1I3), and proteolytic fragments derived from these proteins. Each .alpha.-macroglobulin bound to the murine peritoneal macrophage .alpha.-macroglobulin receptor with comparable affinity (Kd .apprx. 1 nM). A carboxyl-terminal 20-kDa fragment was isolated from each of these proteins, and this fragment bound to .alpha.-macroglobulin receptors with Kd values ranging from 10 to 125 nM. The amino acid identity between the homologous carboxyl-terminal 20-kDa fragments of human and bovine .alpha.2M was approximately 90%, while the overall sequence homology between all carboxyl-terminal fragments studied was 75%. The interchain disulfide bond present in the human .alpha.2M carboxyl-terminal 20-kDa fragment was conserved in bovine .alpha.2M and rat .alpha.1I3, but not in rat .alpha.1M. The clearance of each intact .alpha.-macroglobulin-proteinase complex was significantly retarded following treatment with cis-dichlorodiammineplatinum(II) (cis-DDP). cis-DDP treatment, however, did not affect receptor recognition of purified carboxyl-terminal 20-kDa fragments of these .alpha.-macroglobulins. A carboxyl-terminal 40-kDa subunit, which can be isolated from rat .alpha.1M, bound to the murine .alpha.-macroglobulin receptor with a Kd of 5 nM. Treatment of this subunit with cis-DDP resulted in an increase in the Kd from 5 to 50 nM, similar to the affinity of the isolated carboxyl-terminal 20-kDa fragment of rat .alpha.1M. It is concluded from these studies that the platinum-sensitive component of the .alpha.2M receptor recognition site is located in the carboxyl-terminal region of the protein. However, this region is distinct from the location of the remainder of the .alpha.-macroglobulin receptor recognition site which is contained in the carboxyl-terminal 20-kDa fragment.