The human insulin receptor gene, INSR, and its promoter region have been isolated and characterized. The gene spans >120 kilobase pairs (kbp) and has 22 exons. All introns interrupt protein coding regions of the gene. The 11 exons encoding the a subunit of the receptor are dispersed over >90 kbp, whereas the 11 exons encoding the (3 subunit are located together in a region of =30 kbp. Three transcriptional initiation sites have been identified and are located 276, 282, and 283 bp upstream of the translation initiation site. In addition, a 247-bp fragment from the promoter region possessing 62.6% of the maximal promoter activity has been identified. This promoter-active fragment lacks a TATA-like sequence but has two possible binding regions for the transcriptional factor Spl. Comparison of the exon structure of the tyrosine kinase domain of the INSR with the corresponding regions of the human SRC, ROS, and ERBB2 (NGL) protooncogenes indicates that the exonintron organization of this region has not been well conserved.Insulin initiates a variety of metabolic effects upon binding to a specific receptor on the cell surface (1). The isolation and characterization of cDNA clones encoding the human insulin receptor indicate that the a and f3 subunits of the insulin receptor are derived by proteolytic processing of a common 1382 amino acid preproreceptor (2, 3). The 731-amino acid a subunit (Mr, 135,000) is external to the plasma membrane and contains the insulin-binding region. It is linked by interchain disulfide bonds to the 620-amino acid ,B subunit (Mr, 95,000), which includes a 194-amino acid extracellular domain, a 23-amino acid membrane-spanning segment and a 403-amino acid cytoplasmic segment that has intrinsic tyrosine kinase activity. Both the a subunit and the extracellular region of the 13 subunit are glycosylated.Recent studies have indicated that the synthesis of an abnormal insulin receptor can contribute to the development of non-insulin-dependent diabetes mellitus (4, 5). As a first step in characterizing potentially abnormal genes relevant to diabetes, we have determined the exon-intron organization of the human insulin receptor gene, INSR, and characterized the 5' flanking promoter sequences that regulate its expression.

The isoforms of the a, subunits of voltagedependent Ca2+ channels expressed in human pancreatic islets were identified by using a pair of degenerate oligonucleotide primers and the polymerase chain reaction (PCR) to amplify mRNAs encoding a, subunit-like sequences. The sequences of the PCR products indicate that islets express the heart-type a, subunit as well as a second isoform whose complete sequence has not been previously reported. The sequences of cloned cDNAs encoding the human fl-cell, or neuroendocrine-type, a, subunit indicate that it is composed of 2181 amino acids. It shares 68%, 64%, and 41% identity with the sequences of the a, subunits of rabbit heart, skeletal muscle, and brain, respectively, and is predicted to have a similar structure including four homologous domains composed of six membranespanning segments each. RNA blotting studies indicate that the f-cell-type a, subunit is also expressed in brain as well as in the insulin-producing cell lines RINm5F and flTC-3; however, it could not be detected by RNA blotting in a third cell line, HIT-T15. In situ hybridization studies revealed expression of fl-cell-type a, subunit mRNA in fl cells of rat pancreatic islets, implying that this protein may play a role in the regulation of insulin secretion.

We previously reported the cloning of two distinct somatostatin receptor (SSTR) subtypes, SSTR1 and SSTR2. Although both SSTR1 and SSTR2 bound somatostatin specifically and with high affinity, neither was coupled to adenylyl cyclase, a major cellular effector of somatostatin's actions. Here we report the cloning and functional characterization of a third member of the SSTR family. Human SSTR3 is a protein of 418 amino acids and has 45% and 46% identity with human SSTR1 and SSTR2, respectively. RNA blotting studies showed that SSTR3 mRNA could be readily detected in brain and pancreatic islets. The pharmacological properties of human SSTR3 were characterized by transiently expressing the human SSTR3 gene in COS-1 cells. Membranes from cells expressing human SSTR3 bound the somatostatin agonist [125I]CGP 23996 specifically and with high affinity, with a rank order of potency of somatostatin-28 = CGP 23996 > somatostatin-14 > SMS-201-995. Studies using cells transiently coexpressing the human dopamine D1 receptor and human SSTR3 showed that somatostatin was able to inhibit dopamine-stimulated cAMP formation in a dose-dependent manner, indicating that SSTR3 was functionally coupled to adenylyl cyclase. These results indicate that the diverse biological effects of somatostatin are mediated by a family of receptor with distinct, but overlapping, tissue distributions, unique pharmacological properties, and potentially different functions.

The partial sequence of the human insulin-receptor (hINSR) gene is presented. Using the gene sequence as a guide, we selected pairs of oligonucleotide primers from sites in the introns that flank each exon. These primers allowed each of the 22 exons of the hINSR gene to be amplified in vitro by the polymerase chain reaction. The sequences of the gene and oligonucleotide primers will facilitate studies of genetic variation in the hINSR gene and thereby increase our understanding of the role of this gene in the development of insulin-resistant states and glucose intolerance.