Arginine-135, located at the border between the third transmembrane domain and the second cytoplasmic loop of rhodopsin, is one of the most highly conserved amino acids in the family of G protein-coupled receptors. The effect of mutation at Arg-135 on the ability of rhodopsin to undergo desensitization was investigated. Four mutants, R135K, R135Q, R135A, and R135L, were examined for their ability to be phosphorylated by rhodopsin kinase, to bind arrestin, and to activate the rod cell G protein, transducin (Gt). All of the mutants were phosphorylated, bound arrestin, and were able to activate Gt when reconstituted with 11-cis-retinal. Surprisingly, several of the mutants could be phosphorylated by rhodopsin kinase and could bind arrestin in the absence of 11-cis-retinal but were not able to activate Gt. These observations represent the first demonstration of a mutant G protein-coupled receptor that assumes a conformation able to interact with its G protein-coupled receptor kinase and arrestin, but not with its G protein, in the absence of ligand.

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We have analyzed the distribution of the ~1 isotype of rat protein phosphatase type 1 catalytic subunit in rat salivary glands. Formaldehyde-fixed paraffin sections were reacted with the PP1T1 antibody using an immunohistochemical method. Positive staining occurred in striated ducts of parotid gland. However, the staining reaction was less intense in submandibular gland. Proteins were also prepared from rat salivary glands and subjected to SDS-PAGE, followed by Western blotting analysis with the PP171 antibody. The antibody interacted with protein corresponding to an estimated molecular mass of 36 kDa present in the parotid gland. The staining reaction was considerably weaker with the proteins from submandibular gland.

SUMMARY: Blotting of rat parotid gland proteins separated by SDS-PAGE and transferred to Immobilon transfer membranes revealed that avidin-peroxidase conjugate interacted with bands having estimated molecular weights of 72, 74, and 120 kDa. Even at the lowest concentration of avidin-peroxidase used in the general ABC method (1:2000 dilution), three bands were clearly discernible.The staining reaction of parotid gland proteins was eliminated on preincubating the proteins with native avidin.The staining reaction was markedly reduced with the proteins obtained from submandibular/sublingual glands.