IDENTIFICATION BY POLYMERASE CHAIN-REACTION OF THE G-PROTEIN-COUPLED RECEPTOR KINASES EXPRESSED IN THE MOUSE GONADOTROPIC ALPHA-T3-1 CELL-LINE

G protein-coupled receptor kinases (GRK 1-6) stimulate shortterm desen sitization (<5 min) by phosphorylating G-protein coupled receptors, an d also participate in receptor sequestration, which may relate to inte rmediate-term desensitization (30-60 min). The existence of such kinas es and hence a potential role for them in gonadotrope/GnRH receptor de sensitization was investigated using the PCR to identify GRKs in messe nger RNA (mRNA) from the mouse alpha T3-1 gonadotrope cell line. The 1 50-bp complementary DNAs amplified by PCR from the kinase catalytic do main were cloned and sequenced. Seventeen of 42 clones were receptor k inases based on high nucleotide identities of 85-100% and amino acid i dentities of 97-100% with rat GRK2 and 3, and with human GRK6. Among t he eight GRK3 clones was one differing from rat GRK3 by a single nucle otide and seven differing by six; no amino acid differences resulted f rom the nucleotide differences. Of the five GRK2 clones, one sequence was identical with rat GRK2, but four sequences differed by three nucl eotides and one amino acid. Among four GRK6 sequences, one showed 15 n ucleotide differences from human GRK6 (with no amino acid differences) , and three had 16 nucleotide and one amino acid differences. For each of the three GRKs found, the most closely related isoform is assumed to be the mouse homolog of rat GRK2 and GRK3, and human GRK6, whereas the others are assumed to be previously undescribed isoforms or subtyp es of GRK2, 3, and 6. Immunocytochemical staining using antibodies to GRK2, 3, and 6 confirmed their presence in alpha T3-1 cells. The funct ion of these GRKs in alpha T3-1 cells is unknown, but they may be invo lved in short-term desensitization of the gonadotrope/GnRH receptor or perhaps, more likely, the sequestration of this receptor during inter mediate-term desensitization.