Stimulation, within 1 min after cAMP stimulation, of aggregation-competent Dictyostelium discoideum amebae was found to cause a rapid (within 1 min) modification of the cell's surface cAMP receptor. The modified receptor migrated on SDS PAGE as a 47,000-molwt protein, as opposed to a 45,000-mol-wt protein labeled on unstimulated cells. The length of time this modified receptor could be detected depended upon the strength of the cAMP stimulus: 3-4 min after treatment with 10 -7 M cAMP, cells no longer possessed the 47,000-mol-wt form of the cAMP receptor. Instead, the 45,000-mol-wt form was present. Stimulation of cells with 10 -5 M cAMP, however, resulted in the persistent (over 15 min) expression of the modified receptor. The time course, concentration dependence, and specificity of stimulus for this cAMP-induced shift in the cAMP receptor were found to parallel the cAMP-stimulated phosphorylation of a 47,000-mol-wt protein. In addition, both phenomena were shown to occur in the absence of endogenous cAMP synthesis. The possibility that the cAMP receptor is phosphorylated in response to cAMP stimulation, and the role of this event in cell desensitization, are discussed.

We previously showed that blood outgrowth endothelial cells (BOECs) had a high affinity for polyurethane (PU) covalently configured with cholesterol residues (PU-Chol). However, the molecular mechanisms responsible for this enhanced affinity were not determined. CD47, a multifunctional transmembrane glycoprotein involved in cellular attachment, can form a cholesterol-dependent complex with integrin αvβ3 and heterotrimeric G proteins. We tested herein the hypothesis that CD47, and the other components of the multi-molecular complex, enhance the attachment of BOECs to PU-Chol. Immunoprecipitation studies, of human and ovine BOECs, demonstrated that CD47 associates with integrin αv and integrin β3 as well as G αi-2 protein. The three-fold increase in BOEC attachment to PU-Chol, compared to unmodified PU, was reversed with the addition of blocking antibodies specific for CD47 and integrin αv and integrin β3. Similar results were observed with the addition of methyl-beta-cyclodextrin (MβCD), a known disruptor of the CD47 complex as well as of the membrane cholesterol content, to seeded BOEC or PU-Chol films. Reducing CD47 expression, via lentivirus transduced shRNA, decreased BOEC binding to PU-Chol by 50% compared to control groups. These data are the first demonstration of a role for the CD47 cholesterol-dependent signaling complex in BOEC attachment onto synthetic surfaces.

No abstract