Figure 5.

The aa 347-758 and 1121-1462 regions of GRIP1 are targets for degradation by PKA and CREB. A, Schematic illustrations of the functional domains of full-length GRIP1 (wt) and the GRIP1 truncated or deletion mutants studied in this report. The numbers represent the spanning or deleted (Δ) aa region of each truncated or deletion mutant, respectively. Q-rich indicates a glutamine-rich region; NRB123 indicates mutations in the 3 LXXLL motifs of the NID. B, Western blot analyses of lysates from COS-1 cells transfected with expression plasmids encoding HA-GRIP1 wt or mutant protein (2 μg), CREB (0.5 μg), and PKA-Cα (0.2 μg), where indicated, were performed using anti-HA and anti-GAPDH antibodies. Relative GRIP1 levels shown to the right were calculated from densitometry analyses (ImageJ) of GRIP1 and normalized to GAPDH. Observed downregulation (+) or no change (−) of GRIP1 protein level by PKA/CREB overexpression is also indicated. The results are representative of at least 3 independent experiments.