Evidence that a Cerebellum‐Enriched, Synaptic Junction Glycoprotein Is Related to Fodrin and Resists Extraction with Triton in a Calcium‐Dependent Manner

Abstract

Subcellular fractions from rat cerebellum and other tissues were examined for the presence of a 240K [kDalton] glycoprotein, designated GP-A. GP-A is enriched in cerebellum synaptic junction (SJ) fractions when compared to parent synaptic plasma membrane (SPM) fractions and is not detected in forebrain SPM or SJ fractions. GP-A was not detected in myelin, mitochondria, purified nuclei or cytosolic fractions from cerebellum, but was present in microsomal fractions. GP-A is partially soluble in the non-ionic detergent Triton X-100 and is completely soluble when cerebellum SPM are treated with the ionic detergent N-lauryl sarcosinate. The solubilization of GP-A from cerebellum membranes was a function of bound Ca2+. Pretreating SPM with 100 .mu.M-1mM Ca2+ decreased the solubility of GP-A in Triton .apprx. 3-fold. GP-A is a major concanavalin A (Con A)-binding glycoprotein in cerebellum SJ fractions and migrates on sodium dodecyl sulfate (SDS) gels with a slower relative mobility than the 235K/230K fodrin doublet. Comparisons between purified fodrin and doublet. Comparisons between purified fodrin and the 235K/230K doublet in cerebellum and forebrain synaptic fractions by 2-dimensional peptide mapping indicated they were identical. The Con A-binding property of GP-A was exploited to purify it by affinity chromatography with agarose-Con A. Peptide mapping comparisons between affinity-purified GP-A and GP-A in SPM and SJ fractions indicated that GP-A in synaptic fractions is apparently homogeneous. Peptide map comparisons between GP-A and 235K fodrin polypeptide indicated these 2 synaptic components are highly related (50% of their respective peptides are shared). The 235K fodrin polypeptide in SJ reacted with anti-fodrin antisera on Western blots; GP-A failed to cross-react. GP-A is highly enriched in cerebellum compared to other neuronal and nonneural tissues. GP-A is enriched in SJ relative to SPM fractions, is related to fodrin, and is most likely a cell-surface glycoprotein at asymmetric synapses in cerebellum. GP-A may be involved in neuronal recognition or synaptic transmission in the cerebellum. The important role of Ca in synaptic transmission, together with the decreased solubility of GP-A in Triton that results from micromolar concentrations of Ca, suggests that GP-A may play a role in stabilizing cerebellar synaptic junctions.