Effects of subunit mutation on the rotational dynamics of Fc epsilon RI, the high affinity receptor for IgE, in transfected cells.
Erythrosin-labeled immunoglobulin E (IgE) and time-resolved phosphorescence anisotropy were used to monitor the rotational dynamics of transfected wild-type (alpha beta gamma 2) and four mutant Fc epsilon RI receptors in the monomeric and dimeric state on P815 cells. Erythrosin-IgE bound to Fc epsilon RI on cells transfected with either beta or gamma subunits with truncated COOH-terminal cytoplasmic segments exhibit faster rotational motion than when bound to Fc epsilon RI on cells transfected with wild-type subunits. Deletion of the NH2-terminal cytoplasmic segment of the beta subunit or the COOH-terminal cytoplasmic segment of the alpha subunit does not cause any significant change in the anisotropy decay. Dimers of IgE-receptor complexes formed with anti-IgE monoclonal antibody B1E3 exhibit substantially slower anisotropy decays for all the receptor constructs used, including a receptor construct that only contains the ectodomain of the alpha subunit anchored to Chinese hamster ovary (CHO) cell membranes through a lipid tail. This loss of rotational motion of dimeric IgE-Fc epsilon RI complexes may be due to nonspecific entanglement or to specific interactions involving IgE or the extracellular portion of alpha. The results suggest that the beta and gamma subunits of the tetrameric alpha beta gamma 2 receptor participate in interactions with other membrane components even in the absence of receptor aggregation. The loss of such interactions may be related to the functional impairments previously determined for these mutants.
Chang
E Y
EY
Department of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Mao
S Y
SY
Metzger
H
H
Holowka
D
D
Baird
B
B
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004923:Erythrosine; D018014:Gene Transfer Techniques; D007073:Immunoglobulin E / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D017455:Receptors, IgE / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012399:Rotation
D017455:Receptors, IgE; D007073:Immunoglobulin E; D004923:Erythrosine