For this activity we are going to look at different G proteins receptors (GPCR's) and look for highly conserved areas. These are areas that have not mutated (or if they did the mutation was fatal and selected against) and therefore must be very important!

1. Go to the Biology Workbench and log in.

2.Select Session Tools and then resume session. Click on Protein Tools. This should provide you with your Type 2 angiotensin II receptor for humans sequence from the previous part .

3. Because G protein receptors are proteins we will use the Protein Tools ( Duh!) all day today. We will use the Ndjinn Multiple Database search. Select 3 G proteins from the list below that you would like to compare to the Type 2 angiotensin II receptor for humans. In many cases you may go with the easier name (acetylcholine receptor and not acetylcholine Vertebrate type 3 receptor) to find the protein you want.

4. Use the appropriate names from below, but to each you must add the word "receptor" for your search because we are interested in the cell membrane receptor for the G protein. Select the Swissprot Database. You may only be able to search for one at a time most likely.



5. Select your choice (for humans if possible) and Import Sequences.

6. When you've imported all 3, select all of the sequences plus Type 2 angiotensin II receptor for humans by clicking on the boxes to the left of the names. In the window select CLUSTALW. - (perform selected operation) When the new page appears go to the bottom and (submit). This aligns the sequences. Remember you are looking at amino acid sequences.

7. Can you find Steve's DRY motif that was so important to him?

Question 1 Which of the G proteins that you are looking at had this group?

8. Print out the CLUSTALW that is on your screen. A color printer will help but is not necessary. Look for consensus or conservation between the Type 2 angiotensin II receptor for humans and your proteins. If you have it between Type 2 angiotensin II receptor for humans and 2 of the others note that.

Question 2 Complete the table below based on your alignment. (You may copy and paste this table into word to make it easier.)
(HINT: Each complete row consists of 60 amino acids)

Type 2 angiotensin II receptor for humans	Name of the G protein receptor you are researching	A. Number of Amino Acid Positions conserved (they were the same as the human)	B. Number of Different Amino Acid Positions Non-Conserved areas	Percentage of Conserved Amino Acids =(Column A/ total A+B amino acids *100%)
AT II vs.

9. Look for highly conserved areas that are found in all 4 G protein receptors. We are most interested in the intracellular groups. The data you need is given below. Use Type 2 angiotensin II receptor for humans as your ruler. Its amino acids are numbered 1-363. Dashes do not count. They represent areas where the 2 proteins are not the same in length. The computer lines them up based on the areas that are the same. Put a box around the areas that represent the intracellular loops. Then mark the conserved groups within those boxes.


Question 3 Are there any conserved groups found in the intracellular loops of all 4 of the G protein receptors that you looked at? What are they and where are they found? Example, DRY from amino acid 141 to 143 in the second intracellular loop.

9. Scroll down to the Clustal W Dendogram. This shows how closely relately organisms or proteins are. Using the right click button, copy and paste this into your word document.

Question 4 Explain the evolutionary relationships of the proteins you have worked with.