The role of the Stargazin-like protein gamma 7
Voltage-dependent calcium channel (VDCC) gamma-subunits were first identified by their co-purification with VDCC ai-subunits from skeletal muscle. Since then a further seven gamma-subunits have been identified by their homology to gamma1. Their role however is not limited to interaction with VDCCs. The second gamma-subunit identified, gamma2 (also known as Stargazin), along with gamma3, gamma4 and gamma8 have been shown to be involved in regulation of a-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) receptor trafficking. Also, recent work has shown that expression of the VDCC ai subunit, Cav2.2, is reduced when it is co-transfected with gamma7. The objectives of this study were to investigate the mechanism by which this reduction of Cav2.2 expression takes place and identify the role of gamma7. Several truncated gamma7 subunits were made to examine if potential endoplasmic reticulum (ER) retention motifs, present in the C-terminal region of gamma7, are involved in the reduction of Cav2.2 expression. Truncation of gamma7 from gamma217 (including both potential ER retention (RXR) motifs) did not alter its distribution compared to full length gamma7, both being localised in the ER in transiently transfected COS7 cells. This suggests that these ER retention motifs are not critical in the effect of gamma7 on Cav2.2 expression. However, western blotting experiments, also using transiently transfected COS7 cells, show that the C-terminal region of gamma7 is necessary and sufficient for the reduction of Cav2.2 expression. This was confirmed electrophysiological using a Xenopus oocyte overexpression system to aid in the elucidation of its role binding partners for gamma7 were sought. PC 12 cells, a rat pheochromocytoma cell line, were used a potential source of binding partners. RT-PCR and immunocytochemistry were used to show that gamma7 is endogenously expressed in PC12 cells. Immunoprecipitation of HA tagged gamma7 from a stably transfected PC 12 cell line identified three ribonucleotide binding proteins as potential binding partners.