Cloning, sequencing and proposed structure for a prostaglandin F 2α receptor regulatory protein

Abstract

The cDNA has been cloned for a protein which copurifies with and colocalizes with [ 3H]PGF 2α binding activity, yet differs from the previously cloned prostaglandin F 2α receptor. Polyclonal antibody, produced against one of two protein bands present in a highly purified preparation of [ 3H]PGF 2α binding activity isolated from pregnant bovine corpus luteum, was used to screen a rat ovary cDNA expression library. A single strongly positive clone was identified containing a 4 kilobase (kb) insert. Northern analysis using this cDNA as a probe revealed the expression of a 6 kb mRNA with a tissue distribution similar to that seen by immunohistochemical analysis with the polyclonal antibody. Tissues possessing the largest quantity of the protein's mRNA are reproductive tissues, lung, and heart. Directed cDNA synthesis was required to clone the 5′ end of the cDNA. Verification that the correct cDNA was cloned is provided by alignment of the predicted protein's mature amino terminal amino acid sequence with sequence observed by protein sequencing. Translation of the predicted 879 amino acid open reading frame (ORF) suggests a protein structure exhibiting six glycosylated immunoglobulin type loops, one of which may either be membrane associated or may be the site of association with another protein, a transmembrane region, and a short, highly charged, carboxy terminal cytoplasmic tail. Based upon searches of the NIH and EMBL protein databanks, this is a unique protein. The FPRP mRNA is notable for a highly structured G,C rich 5′ end and over 3 kb of 3′ untranslated region (UTR) that includes 7 ATTTA ‘destabilization sequences’ and an ‘inflammatory mediator’-like sequence. These characteristic sequences in the 3′ UTR suggest that the mRNA is tightly regulated and may code for a protein that is functionally related to an inflammatory mediator. Functional studies in the accompanying report suggest a negative regulatory function for this protein. We suggest the name prostaglandin F 2α receptor regulatory protein (FPRP).