Cloning and functional characterization of adhesion molecules in the nervous system of Helix pomatia

A large number of proteins belonging to the IgCAM family are involved in various aspects of neuraldevelopment and partecipate in the regulation of synaptic formation. We focused on the mouseneuronal glycoprotein F3/contactin, a member of the immunoglobulin superfamily that has beenimplicated in several functions in the vertebrate nervous system, including axonal growth,pathfinding and synaptic activity.In previous studies, by using the anti mouse F3/contactin antisera, we found that three differentcomponents were expressed in the Helix pomatia nervous system and we investigated theirdistribution on Helix pomatia ganglia and on cultured Helix neurons. We also demonstrated thatthese molecules appeared to be important for cell adhesion and neurite outgrowth of neurons inculture and that they were involved in the target dependent regulation of neurotransmitter releasefrom presynaptic terminals. We called these protein components Helix F3/contactin Related Proteins(HFRPs).In the present study, by screening an expression library obtained from the Helix pomatia nervoustissue with the mouse F3/contactin antibody, we cloned two related proteins called HFRP1andHFRP2.Alignment analysis showed that these two molecules shared almost the same sequence andstructure between by the signal peptide and the first FNIII domain, but after that they remarkablydifferentiate. By northern blotting analysis, in which the common coding sequence was used asprobe, we identified a single 6.3 Kb mRNA,suggesting that these isoforms could arise fromalternative splicing of a single gene, and by CHO transfection we demonstrated that either HFRP1than HFRP2showed a clear surface distribution. Microinjected isolated neurons with an antisensemRNAgenerated from HFRP2to inhibit the protein expression showed a massive reduction ofneurite extension and branching, suggesting that HFRPs are surface proteins involved in modulationof neuronal growth.