Cloning of Expressed Neurotrimin Defines a New Subfamily Of
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The Journal of Neuroscience, March 1995, 15(3): 2141-2156 Cloning of Neurotrimin Defines a New Subfamily of Differentially Expressed Neural Cell Adhesion Molecules Arie F. Struyk,’ Peter D. Canoll,2 M. J. Wolfgang,’ C. L. Rosen,’ Peter D’Eustachio,s and James L. Sajzer’+ Departments of ‘Cell Biology, 2Pharmacology, 3Biochemistry, and 4Neurology, New York University Medical School, New York, New York 10016 Previous studies in the laboratory indicated that glycosyl- that could be important in the specification of neuronal phosphatidylinositol (GPI)-anchored proteins may generate connectivity. diversity of the cell surface of different neuronal popula- [Key words: glycosylphosphatidylinositol (GPI), neurotri- tions (Rosen et al., 1992). In this study, we have extended min, opiate binding-cell adhesion molecule (OBCAM), in these findings and surveyed the expression of GPI-an- situ hybridization, neural cell adhesion molecules, chro- chored proteins in the developing rat CNS. In addition to mosome mapping] several well characterized GPI-anchored cell adhesion mol- ecules (CAMS), we detected an unidentified broad band of A striking feature of the CNS of higher vertebrates is the ex- 65 kDa that is the earliest and most abundantly expressed quisite precision with which functionally appropriateneural cir- GPI-anchored species in the rat CNS. Purification of this cuits are establishedduring development. Neuronsthat residein protein band revealed that it is comprised of several related widely separatedareas extend processesthat must successfully proteins that define a novel subfamily of immunoglobulin- navigate distinct cellular environments en route to their synaptic like (lg) CAMS. One of these proteins is the opiate binding- targets. The precision observed in the formation of neuronalpro- cell adhesion molecule (OBCAM). We have isolated a cDNA jections is thought to result from mechanismsthat regulate encoding a second member of this family, that we have growth cone pathfinding and target recognition, followed by lat- termed neurotrimin, and present evidence for the existence er refinement and remodeling of theseprojections by events that of additional family members. Like OBCAM, with which it require neuronal activity (Goodman and Shatz, 1993). The abil- shares extensive sequence identity, neurotrimin contains ity of nerve fibers to make specific pathfinding choices during three immunoglobulin-like domains. Both proteins are en- development and to select their appropriate synaptic target, coded by distinct genes that may be clustered on the prox- therefore, underliesthe precisepatterns of neuronal connectivity imal end of mouse chromosome 9. Characterization of the observed in the mature nervous system. Emerging evidence in- expression of neurotrimin and OBCAM in the developing dicates that different neurons extend nerve fibers that are bio- CNS by in situ hybridization reveals that these proteins are chemically distinct and rely on specific guidance cues provided differentially expressed during development. Neurotrimin by cell-cell, cell-matrix, and chemotropic interactions to reach is expressed at high levels in several developing projection their appropriate synaptic targets (Goodman and Shatz, 1993). systems: in neurons of the thalamus, subplate, and lower One of the means by which diversity of the neuronal cell cortical laminae in the forebrain and in the pontine nucleus, surface may be generated is through differential expression of cerebellar granule cells, and Purkinje cells in the hindbrain. cell surface proteins referred to as cell adhesion molecules Neurotrimin is also expressed at high levels in the olfactory (CAMS). Neuronally expressedCAMS have been implicated in bulb, neural retina, dorsal root ganglia, spinal cord, and in diverse developmentalprocesses, including migration of neurons a graded distribution in the basal ganglia and hippocam- along radial glial cells, providing permissive or repulsive sub- pus. OBCAM has a much more restricted distribution, be- stratesfor neurite extension, and in promoting the selective fas- ing expressed at high levels principally in the cortical plate ciculation of axons in projectional pathways (reviewed in Jessel, and hippocampus. These results suggest that these pro- 1988; Edelman and Crossin, 1991). Interactions between CAMS teins, together with other members of this family, provide present on the growth cone membraneand moleculeson appos- diversity to the surfaces of different neuronal populations ing cell membranesor in the extracellular matrix, are thought to provide the specific guidance cues that direct nerve fiber out- Received Aug. 1, 1994; revised Sept. 13, 1994; accepted Sept 15, 1994. growth along appropriate projectional pathways. These interac- We are grateful to Pat Levitt for sharing unpublished sequence data and, tions are likely to result in the activation of various secondmes- together with John Pintar and Marla Luskin, for helpful discussions about the sengersystems within the growth cone that regulate neurite out- in situ hybridization studies; Bill Lane at the Harvard Microchemical facility for advice on preparation of proteins for microsequencing, and M. Low for his growth (Doherty and Walsh, 1992). gift of PIPLC. This work was supported by a grant from the American Paralysis In higher vertebrates, most neural CAMS have been found to Association and the NIH (NS 26001) to J. Salzer. A. Struyk and C. Rosen are be members of three major structural families of proteins: the Medical Scientist Trainees supported by NIH training grant 5T32 GM07308 from NIGMS. J. Salzer is a recipient of an Irma T Hirsch1 Career Scientist integrins, the cadherins, and the immunoglobulin gene super- Award. family (IgSF) (Jessel,1988; Takeichi, 1990; Reichardt and To- Correspondence should be addressed to Dr. James L. Salzer, Department of Cell Biology, New York University Medical School, 550 First Avenue, New maselli, 1991). Cell adhesion molecules of the IgSF (or Ig- York, NY 10016. CAMS), in particular, constitute a 1al;gefamily of proteins fre- Copyright 0 1995 Society for Neuroscience 0270-6474/95/152141-16$05.00/O quently implicated in neural cell interactions and nerve fiber 2142 Struyk et al. - Cloning and Expression of Neurotrimin outgrowth during development (Salzer and Colman, 1989; viously described procedures (Rosen et al., 1992). Tissue was homog- enized with either a Dounce or Polytron homogenizer in 10 mllmg wet Briimmendorf and Rathjen, 1993). In some instances, members wt in calcium-free PBS containing 1 mu EDTA and the protease in- of the IgSF are believed to function in pathfinding choices. For hibitors antipain, pepstatin A, and leupeptin, all at a concentration of instance, TAG-l is expressed in a region where clearly defined 0.1% (PE buffer). The homogenate was spun first at 700 X g for 10 pathfinding decisions are being made such as on commissural min to remove unbroken cells and then at 12,000 X g for 1 hr at 4°C. axons of the spinal cord before crossing the midline (Dodd et The resultant pellet was washed twice in PE buffer at 4”C, resuspended in 1 ml, and the protein concentration was determined by the Micro- al., 1988). Fasciclin II is critical for the formation of the MPI BCA method following the manufacturer’s instructions (Pierce). Mem- fascicle in both grasshopper and Drosophila (Grenningloh et al., brane protein (200 pg) was pelleted in a microfuge and resuspended in 1991). However, the majority of mammalian Ig-CAMS appear calcium-free PBS containing NHS-sulfa-biotin (Pierce) at a concentra- to he too widely expressed to specify navigational pathways or tion of 0.5 mglml. Samples were incubated for 30 min at 4”C, after synaptic targets suggesting that other CAMS, yet to be identified, which the membrane fractions were pelleted once more and washed once in Dulbecco’s MEM, and three times in calcium-free PE buffer. have a role in these more selective interactions of neurons. Pelleted membrane proteins were either used immediately or frozen at Of the recently identified neural Ig-CAMS, many have been -80°C for later use. Subsequent purification of GPI-anchored proteins found to be attached to the plasma membrane via a glycosyl- by release from a Triton-X-114 detergent phase into the aqueous phase phosphatidylinositol (GPI) anchor. Thus, the vertebrate neural by PIPLC treatment and subsequent precipitation with TCA was carried proteins TAG-l/axonin-1, F3/Fll, Thy-l, and the 120 kDa is- out as previously described (Rosen et al., 1992). The precipitated proteins were fractionated by SDS-PAGE and elec- oform of NCAM (Tse et al., 1985; He et al., 1986; Gennarini et troblotted onto nitrocellulose, which was then blocked with PBS con- al., 1989; Furley et al., 1990) and the invertebrate proteins fas- taining 0.5% Tween-20, 150 mu NaCI, 10% glycerol, 1 mu D-ghCOSe, ciclin I, amalgam, lachesin, and apCAM all exhibit GPI-an- 3.5% BSA, and 2.5% fat-free dried milk for 1 hr at RT To detect the chorage (Seeger et al., 1988; Hortsch and Goodman, 1990; May- GPI-anchored proteins, blots were then incubated with either lZ51-strep- tavidin or alkaline phosphatase-conjugated streptavidin (Bio-Rad) for 1 ford et al., 1992: Karlstrom et al., 1993; reviewed in Salzer et hr. The blots were rinsed extensively in 0.5% Tween-20 in PBS, and al., in press). In addition, a number of studies have implicated exposed for autoradiography or developed with the BCIP-NPT substrate GPI-anchored proteins in providing specific guidance cues dur- (Keirkegaard and Perry), respectively.