Ras-associating domain proteins: a new class of cyclic nucleotide-gated channel modulators

Abstract

The Ras is a protein subfamily of small GTPases that are involved in cellular signal transduction. Members of Ras family are all related in structure and regulate diverse cell behaviors. Ras-associating/binding (RA/RBD) domain containing proteins perform several different functions ranging from tumor suppression to being oncoproteins. Their role in different biological processes may be unclear and highly divergent but what is clear is that they convergently function by interacting with Ras proteins through their RA/RBD subdomains directly or indirectly. Apart from interacting with Ras proteins, there is no perceptible relationship between these proteins or their highly unrelated protein bodies. The heterogeneity among these RA domains allows them to interact with Ras proteins of different types as well as several other proteins which contain similar motifs. Very recently we have demonstrated that growth factor receptor bound protein 14 (Grb14) RA-domain binds to photoreceptor cyclic nucleotide-gated channel (CNG) and inhibits its activity in vivo. In this study we have examined two other RA domain containing protein phosphates expressed in retina, PHLPP1 and PHLPP2 on CNG channel activity. Our data indicate that not all RA domain proteins are modulators of CNG channel, suggesting the existence of heterogeneity among several RA domain proteins.

Fig. XX.1

Comparative domain analysis of the Grb14 with PHLPP1 (β) and PHLPP2. A. The primary structure analysis of the PHLPP1 (β) and PHLPP2 shows that these proteins contain RA and PH domain in a pattern similar to the Grb14 family. Various domains of the proteins are shown diagrammatically. B. The primary structural homology between RA domains of these proteins was established by multiple sequence alignment. The similarity consensus is 90% (red: Grb14 and PHLPP1 (β) and 2; Blue: Grb14 and either PHLPP1 (β) or PHLPP2). Pro, proline rich region; RA, Ras-associating; PH, pleckstrin homology; BPS, between PH and SH2; SH2, Src-homology-2 region; LRR, leucine-rich repeat; PP2C, Protein Phosphatase 2C; PDZ, post synaptic density protein (PSD95), drosophila disc large tumor suppressor (DlgA), and zonula occludens-1 protein (zo-1).

Fig. XX.2

Effect of PHLPP1 and PHLPP2 on CNG channel activity. A. Myc- CNGA1 was transiently co-expressed with myc-Grb14, FLAG-PHLPP1 or FLAG-PHLPP2 and examined for their effect on channel activity. Cells transfected with Grb14, PHLPP1 (β) or PHLPP2 alone were used to measure the non-channel mediated Ca²⁺ permeability. CNGA1 co-transfected with empty plasmid was used as control. B. Lysates were subjected to immunoblot analysis with anti-Myc antibodies for CNGA1 and anti-FLAG antibodies for PHLPP1 and PHLPP2 respectively. Data mean ± SD, n=3; p < 0.05.