Pyk2 Binds b1 Integrin After Integrin Activation in Neuronal Cultures

One of the most common types of orofacial pain is caused by temporomandibular joint (TMJ) disorder. The mechanisms of orofacial pain are poorly understood. Opioids, such as morphine derivatives are often used in dentistry for the treatment of orofacial pain. Previous results have shown that integrin receptors can modulate the affinity of opioid receptors for ligands, but it is not known how the signal from a stimulated integrin is transduced to the opiod receptor. Here, we bound blocking b1 antibodies to neuron cells. An integrin is any of a family of heterodimeric cell-adhesion receptors, consisting of two noncovalently linked polypeptide chains, designated alpha and beta. Objective: Does signal transduction from stimulated integrins occur via the protein pyk2 pathway, as judged by formation of a b1-pyk2-caveolin-1 supramolecular complex? Methods: Male rat trigeminal ganglion neuronal cultures (8 DIC) were stimulated with blocking b1 antibodies and the supramolecular complex was immunoprecipitated with antibodies against pyk2 and protein G agarose. Precipitated proteins were recovered from the antibody-agarose complex using low and high NaCl concentrations and analyzed by SDS-PAGE/ Western blot. Results: Supramolecular complexes isolated from cells treated with b1 blocking antibodies contained 7.9x and 2.4x more b1 in low (0.15M) and high (0.5M) NaCl extracts, respectively, than untreated cells, and 24.6x and 2.4x more caveolin-1 in low and high NaCl extracts, respectively, than untreated (control) cells. Conclusion: Stimulation of integrins with b1 blocking antibodies results in a high affinity binding of b1 and caveolin-1 to pyk2, suggesting that the signal transduction pathway that leads to opioid receptor modulation involves the pyk2 pathway. This study was supported by UTHSCSA- Hispanic Center of Excellence- Dentistry, NIDCR grant P01DA016719, and NIDCR, T-32 Grant #DE14318- CO STAR Program.