The murine P84 neural adhesion molecule is SHPS-1, a member of the phosphatase-binding protein family - PubMed (original) (raw)

The murine P84 neural adhesion molecule is SHPS-1, a member of the phosphatase-binding protein family

S Comu et al. J Neurosci. 1997.

Abstract

P84 is a neuronal membrane glycoprotein that promotes the attachment and neurite outgrowth of cultured murine cerebellar cells. The heterophilic adhesive properties of P84 and its localization at sites of synaptogenesis suggest that it may be involved in regulation of synapse formation or maintenance. P84 is expressed in subsets of neurons throughout the CNS. By cloning the cDNA encoding murine P84, we have discovered that this molecule is a member of a family of phosphatase-binding proteins and is identical to the murine SHPS-1 cDNA. Here we report the cloning of two alternatively spliced forms of P84 and describe its localization within the CNS by in situ hybridization.

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Figures

Fig. 1.

Fig. 1.

A, Diagram of large and small forms of P84 cDNA. The exon corresponding to bases 461–1114 is removed by alternative splicing to generate the P84 small mRNA. The protein coding region is shown as a shaded box, whereas the signal peptide (SP) and transmembrane domain (TM) are indicated as black boxes. The positions of Bam_HI (B) and_Xba_I (X) sites within the 3′-untranslated region are shown. The segment of the cDNA that was used to prepare RNA probes for in situ hybridization is shown as a hatched box over the corresponding_Bam_HI and Xba_I sites. B, The translated peptide sequence corresponding to P84 large mRNA. The peptide segment that is removed by splicing (shown in A) is located between the arrowheads. Four potential Tyr phosphorylation sites and one possible Thr phosphorylation site in the cytoplasmic domain are shown by shadowing. Potential N-glycosylation sites in the extracellular domain are shown as_bold and underlined characters. The signal peptide and the single transmembrane domain are indicated by_underlining, whereas two Pro-rich segments are indicated by heavy underlining. The N-terminal peptide fragment and the internal tryptic peptide fragment that were sequenced are_boxed_.

Fig. 2.

Fig. 2.

Deglycosylation of P84. Lanes A,C, The 86 and 77 kDa bands purified by affinity chromatography and electrophoresis. Lanes B,D, The same peptides treated with_N_-glycosidase F. Apparent shifts in molecular weight from 86 to 64 and from 77 to 42–55 kDa are observed. Molecular weights in kilodaltons are indicated on the left.

Fig. 3.

Fig. 3.

P84 sequence homologies. A–C, Alignment of the first (A), second (B), and third (C) Ig-like domains of P84 with Ig-κ light chain V region, Ig-λ C region, and Ig-γ CH1 region, respectively. In each case the P84 sequence is shown on the top line, and amino acid identities are indicated_below_, with conservative substitutions indicated by_+_. Conserved Cys (C) and Trp (W) residues are boxed.D, Alignment of the cytoplasmic domain with segments of the mouse and human IRS-1 peptide. The segments of mouse IRS-1 are not contiguous and are similar to regions of P84 around Thr and Tyr phosphorylation sites. Also shown here is the similarity to a short Pro-rich segment of human IRS-1. E, Alignment of the transmembrane and juxtamembrane segments of P84 with receptor protein tyrosine phosphatase α.

Fig. 4.

Fig. 4.

Left, Northern blot of cerebellum (lane 1) and cerebrum (lane 2) from P15 mouse probed with P84 cDNA demonstrates a thick band just below the 28S rRNA marker (corresponding to ∼4 kb). The blot was prepared by electrophoresis of 10 μg of total RNA per lane. Right, P84 expression in neural and non-neural tissues. Northern blot prepared with RNA (10 ug per lane) from liver (lane 1), kidney (lane 2), skeletal muscle (lane 3), heart (lane 4), spinal cord (lane 5), cerebrum (lane 6), and cerebellum (lane 7). This represents an overexposed autoradiogram, showing faint bands in liver, kidney, and heart. _Arrowheads_and arrows correspond to 28 and 18 S rRNA bands.

Fig. 5.

Fig. 5.

Detection of large and small forms of P84 mRNA in different tissues by RT–PCR. cDNA pools from cerebellum (lane 1), cerebral cortex (lane 2), spleen (lane 3), thymus (lane 4), liver (lane 5), skeletal muscle (lane 6), heart (lane 7), and spinal cord (lane 8) were amplified with the U36 and L647 primers. Lane 9 is a positive control reaction done with the full-length (large) P84 cDNA as template, and lane 10 is a positive control using SC500 plasmid DNA (partial small cDNA) as template. To the left of lane 1 is a 1 kb ladder.

Fig. 6.

Fig. 6.

Immunohistochemical localization of P84 in cerebellum. Ultracryomicrotome sections showing intense P84 staining in the molecular layer (A–C) and in synaptic glomeruli (ring-shaped structures indicated by arrows in_B_ and C). Weak staining is observed on granule cell bodies. pkj, Purkinje cells; other abbreviations are defined in the Figure 7 legend. Scale bars: A, 200 μm;B, 50 μm; C, 30 μm.

Fig. 7.

Fig. 7.

P84 mRNA distribution in the cerebellum.A, P84 in situ hybridization of a parasagittal section of adult cerebellum. Prominent expression is detected in the granule cell layer (gr), and no signal is observed in the molecular layer (mol) or white matter (wm). Particularly strong expression is observed in a layer immediately below the layer of Purkinje cells.B, A higher magnification of the cerebellar cortex. Here, the strong signal in the thin cytoplasm of granule cells is apparent (white arrow). The Purkinje cells (black arrowheads) are unstained, whereas a strong layer of staining just below the Purkinje cells is indicated with open arrows. Scale bar: B, 40 μm.

Fig. 8.

Fig. 8.

P84 mRNA distribution in selected brain regions.A, A parasagittal section of hippocampus. Note the prominent staining of granule cells of the dentate gyrus (d) and the absence of staining at the CA1–CA3 border, indicated by the_open arrow_. B, A parasagittal section of the caudal portion of neocortex. Note the laminar variation in P84 expression with particularly strong expression in layer IV.p, Pia; s, subiculum. C, A parasagittal section of olfactory bulb. m, Mitral cell layer; pg, periglomerular cells; epl, external plexiform layer; and g, granule cells. Scale bar, 300 μm.

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