Evidence for a second alpha 2-macroglobulin receptor.

U.K Misra,C.T Chu,G Gawdi,S.V Pizzo

doi:10.1016/s0021-9258(18)99909-6

Abstract

alpha 2-Macroglobulin (alpha 2M)-methylamine binds to purified low density lipoprotein receptor-related protein (LRP), and it is assumed that LRP functions as the alpha 2M receptor in vivo. Binding of alpha 2M-methylamine to macrophage receptors elevates intracellular calcium ([Ca2+]i), inositol phosphates, and cyclic AMP. We have employed human alpha 2M-methylamine and recombinant receptor binding fragment (RBF) to study transduction mechanisms. Macrophages exposed to either ligand demonstrated a rapid rise in [Ca2+]i. Since the 39-kDa LRP/alpha 2M receptor-associated protein (RAP) blocks alpha 2M binding to LRP, we explored the effects of RAP upon signaling. Pretreatment of macrophages with RAP did not block the increase in [Ca2+]i elicited by alpha 2M-methylamine or RBF, suggesting a distinct binding site. RBF also elicited a transient 1.5-2.0-fold increase in inositol 1,4,5-triphosphate. In permeabilized macrophages, GTP gamma S and Gp-p(NH)p potentiated and sustained this inositol 1,4,5-triphosphate increase. Preincubation of permeabilized macrophages with GDP beta S abrogated the effects of GTP gamma S. Our results suggest that the signaling alpha 2M receptor is coupled to a pertussis toxin-insensitive G protein and possibly to a cholera toxin-sensitive G protein. We conclude that macrophages contain a second alpha 2M receptor that is G protein-coupled.

Highlights

Macrophages exposed to either ligand demonstrated a rameric a-macroglobulins
We have ceptor is coupled to a pertussis toxin-insensitivGe pro- shown thatexposure of murine peritoneal macrophages to hutein and possibly to a cholera toxin-sensitiGveprotein. man a,M-methylamine elevates intracellular calcium ([Ca“],), We conclude that macrophages contain a second a,M inositol phosphates, andcyclicAMP (Misra et al.,1993)
Macrophages cultured lecting backgrounddata (3-5 min), the receptor bindingfragment (RBF) (40nM), a,M-methylamine overnight were washedtwice with HHBSS, preincubated ian volume of (40 KIM)and RAP (200 n ~w)ere added alone or in various combinationsHHBSS for 5 min at 37 "C in 5%CO, and treated with either buffer or to the coverslip

Summary

EXPERIMENTAL PROCEDURES

(thi0)triphosphate;GDPPSg, uanyl-5’-yl thiophosphateG; pp(NH)p, Reagents-Sterile distilled water was obtained from Abbott Laboraguanosine 5’-(P-y-imido)triphosphateI;P,, inositol 1,4,5-triphosphate; tories The ages Exposed to RBF-2-[3H]myo-Inositol-labeledTG-elicited macrophdishes were washed three times with HHBSS to remove nonadherent ages incubated overnight in RPMI 1640 medium were permeabilized cells. Macrophages cultured lecting backgrounddata (3-5 min), the RBF (40nM), a,M-methylamine overnight were washedtwice with HHBSS, preincubated ian volume of (40 KIM)and RAP (200 n ~w)ere added alone or in various combinationsHHBSS for 5 min at 37 "C in 5%CO,, and treated with either buffer or to the coverslip. Monolayers The ADP-ribosylation reaction was initiatedby the addition of either were washed five times with HHBSS containing 10 m LiCI, 1 cholera toxin(1pg/ml, preactivated with m2M0 dithiothreitol for 30 min. The cells wereexposed to buffer, RBF (40 at 30 "C) or pertussis toxin (1pg/ml, preactivated with 40 m dithionM), a,M-methylamine (40 KIM),or RAP (200 KIM)in separate experi- threitol for 20 min) and[32PlNAD(1pCi/30 d)in separate experiments. The mobilization of [Ca2+],after ligand binding generally involves activation of inositol phospholipid-specific

RESULTS

Findings

DISCUSSION