Loop 6 of RhoA confers specificity for effector binding, stress fiber formation, and cellular transformation

Rho family GTPases regulate multiple cellular processes, including cytoskel etal organization, gene expression, and transformation. These effects are a chieved through the interaction of GTP-bound proteins with various downstre am targets. A series of RhoA/Rad and Rho/Ras chimeras was generated to map the domain(s) of RhoA involved in its association with two classes of effec tor kinase, represented by PRK2 and ROCK-I, Although the switch 1 domain wa s required for effector binding, the N terminus of Rho (residues 1-75) was interchangeable with that of Rac, This suggested that the region of Rho tha t confers effector binding specificity lay further C-terminal, Subsequent s tudies indicated that the "insert domain" (residues 123-137), a region uniq ue to Rho family GTPases, is not the specificity determinant. However, a de terminant for effector binding was identified between Rho residues 75-92, R ac to Rho point mutations (V85D or A88D) within loop 6 of Rac promoted its association with PRK2 and ROCK, whereas the reciprocal Rho(D87V/D90A) doubl e mutant significantly reduced effector binding capacity. In vivo studies s howed that microinjection of Rac(Q6IL/V85D/ A88D) but not Rac(Q6IL)) induce d stress fiber formation in LLC-PK epithelial cells, suggesting that loop 6 residues conferred the ability of Rac to activate ROCK. On the other hand, the reciprocal Rho (Q6IL/D87V/D90A) mutant was defective in its ability to transform NM 3T3 cells. These data suggest that although Rho effecters can utilize a Rho or Rac switch I domain to sense the GTP-bound state of Rho, unique residues within loop 6 are essential for determining both effector b inding specificity and cellular function.